Syndecan peptides and polypeptides as inhibitors of vascular endothelial growth factor receptor-2 (vegfr2) and very late antigen-4 (vla-4)

ABSTRACT

The disclosure provides for peptides from syndecan 1 and methods of use therefor. These peptides can inhibit VLA-4 interaction with VEGFR2, thereby preventing tumor cell growth and tissue invasion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/043,951, filed on Feb. 15, 2016 and issued as U.S. Pat. No. 9,878,007on Jan. 30, 2018, which claims the benefit of U.S. Provisional PatentApplication No. 62/119,466, filed on Feb. 23, 2015. Each of theseapplications is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA139872 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND I. Field

This disclosure relates to regulation of cell growth, and moreparticularly to regulation of cancer cell growth. In particular,peptides and polypeptides derived from particular regions of Syndecan 1have been shown to inhibit activation of VLA-4 (α4β1 integrin) andengagement of VLA-4 by VEGFR2, thereby limiting tissue invasion.

2. Related Art

Multiple myeloma, a disease in which malignant plasma cells formdisruptive bone tumors, is the second most prevalent hematologicmalignancy in the United States (Laubach et al., 2010). The emergence ofnew therapies (e.g., bortezomib, thalidomide) has greatly improvedsurvival rates in patients with myeloma (Laubach et al., 2010). However,these therapies slow rather than cure the disease and patients developresistance and become refractory over the course of treatment. Thus, theneed for novel therapies that prevent the progression of the disease andmaintain patient quality of life remains a high priority. A betterunderstanding of the mechanisms involved in disease progression mayidentify new and effective targets for such therapies.

Heparanase (HPSE), an endo-β-D-glucuronidase that degrades heparansulfate (HS) chains on proteoglycans, is a tumor promoter in multiplemyeloma, as well as in many other cancers (Barash et al., 2010, Kelly etal., 2003 and Vlodavsky et al., 2002). HPSE cleaves at highly specificsites within HS chains, releasing biologically active fragments 5 to 7kDa in size that bind and promote the activity of heparin-binding growthfactors. However, HPSE has far-reaching effects beyond the release of HSfragments, including altering the expression of genes that affect theproliferation, invasion and survival of tumor cells and other cells inthe tumor microenvironment (Vlodavsky et al., 2002 and Levy-Adam et al.,2010). A major target of HPSE in multiple myeloma is syndecan-1 (Sdc1,CD138), one of a family of cell surface heparan sulfate proteoglycansfound on most cells. Sdc1 is highly expressed on malignant plasma cellsand has a causal role in multiple myeloma (Khotskaya et al., 2009;O'Connell et al., 2004; Sanderson and Yang, 2008 and Yang et al., 2002).Cells expressing high levels of Sdc1 exhibit enhanced invasion intocollagen gels in vitro and as tumors in vivo (Yang et al., 2002). Incontrast, suppression of Sdc1 expression causes apoptosis in myelomacells (Khotskaya et al., 2009 and Wu et al., 2012).

Induction of metalloproteinase-9 (MMP-9) expression by HPSE, along withits pruning of the HS chains on Sdc1, causes MMP-9-mediated shedding ofSdc1 ectodomain into the tumor microenvironment where the proteoglycanenhances angiogenesis and is likely to have roles in myeloma celladhesion, proliferation and survival (Yang et al., 2007; Mahtouk et al.,2007; Purushothaman et al., 2008 and Purushothaman et al., 2010).(Ramani et al., JBC, 287: 9952-9961 (2012). Indeed, high levels of shedSdc1 in serum correlate with poor prognosis in multiple myeloma (Seidelet al., 2000 and Scudla et al., 2010). Although Sdc1 is shed, thesteady-state level of functional Sdc1 at the cell surface remainsunchanged due to an HPSE-induced increase in receptor expression (Yanget al., 2007; Mahtouk et al., 2007 and Ramani et al., 2012). Thus, theSdc1 exists in at least two functional states in myeloma—a cell surfacereceptor and a bioactive agent in the extracellular milieu—andunderstanding its roles in these states appear highly critical forunderstanding the causes of highly malignant myeloma.

As a cell surface receptor, Sdc1 has an emerging role as an organizer ofintegrin and growth factor receptor signaling. The best-characterizedexample involves the insulin-like growth factor-1 receptor (IGF-1R) andthe αvβ3- or αvβ5-integrin. These receptors are captured by an activesite in the extracellular domain of Sdc1 (aa 92-119 in mouse Sdc1,93-120 in human Sdc1); their capture by Sdc1 at sites of matrix adhesionpromotes activation of the IGF-1R, which generates an inside-out signalthat activates the integrins (Beauvais et al., 2009; Beauvais andRapraeger, 2010; and McQuade et al., 2006). A peptide that mimics theactive site in human Sdc1 (synstatin 93-120, also called SSTN_(IGF1R))disrupts the assembly of this complex on tumor cells and activatedvascular endothelial cells, blocks tumor growth and tumor-inducedangiogenesis, and is a candidate for therapeutic intervention in humandisease. These findings suggest that Sdc1, either as a cell surfacereceptor, or when shed from the cell surface, has a role in activatingreceptor tyrosine kinases and/or integrins.

VLA-4 (very late antigen-4, or the α4β1 integrin) participates in theinfiltration of leucocytes and lymphocytes (Alon and Feigelson, 2002;Alon et al., 1995). In addition to using VLA-4 for extravasation fromthe blood stream, myeloma cells rely on VLA-4 to engage bone marrowstromal cells, and fibronectin (FN) in the marrow extracellular matrix(ECM) for growth and survival (Sanz-Rodriguez et al., 1999; Vande Broeket al., 2008) and to resist therapeutic drugs (Noborio et al., 2009)(e.g., “cell adhesion-mediated drug resistance (CAM-DR)” (Meads et al.,2008; Damiano et al., 2000; Damiano et al., 1999; Schmidmaier et al.,2006). Binding to VCAM-1 on marrow stromal cells also causes release ofMIP-1α and MIP-1β, activating osteoclasts and bone erosion (Abe et al.,2009; Michigami et al., 2000).

Angiogenesis and lymphangiogenesis also play important roles in tumorgrowth and metastasis by providing nutrients exchange as well as avenuesfor tumor cell extravasation, including in hematological malignancies(Orpana and Salven, 2002). VLA-4 expression is required for angiogenesisby both vascular and lymphatic endothelial cells and its activity isespecially prominent in tumors (Garmy-Susini et al., 2013; 2010; 2005).Its matrix ligand, FN, is deposited within the growing vascular andlymphatic microvessels and VCAM-1 is prominently expressed on muralcells (pericytes) that support vascular endothelial cells.

Vascular endothelial cells also rely on vascular endothelial growthfactor receptor-2 (VEGFR2) and this receptor tyrosine kinase is oftenaberrantly expressed in many tumors as well, including in multiplemyeloma (Kumar et al., 2003 and Ria et al., 2003). VEGFR2 inhibitorshave been shown to block proliferation and migration of patient-derivedmyeloma cells (Martinelli et al., 2001). Bone marrow angiogenesisinvolving VEGFR2 also plays an important role in the progression ofmultiple myeloma as in other hematological malignancies (Rajkumar etal., 2000; Vacca et al., 1994 and Rajkumar et al., 2002). Interestingly,Sdc1 extracellular domain shed from myeloma cells expressing high levelsof HPSE has been shown to promote VEGF-dependent angiogenesis in vitro.This depends on its HS chains, but also on its core protein, suggestingthe presence of one or more active sites responsible for the bioactivityof the shed Sdc1.

SUMMARY

Thus, in accordance with the present disclosure, there is provided anisolated and purified peptide segment consisting of between 12 and 100amino acid residues and comprising residues 210-221, 220-236, 210-233,210-236, 214-236 or 214-240 of SEQ ID NO:1. The peptide may be 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 70,75, 80, 85, 90, 95 or 100 amino acid residues in length. The peptide maybe between 12 and 50 amino acid residues in length. The peptide may bebetween 23 and 50 amino acid residues in length. The peptide may bebetween 16 and 30 amino acid residues in length. The peptide may bebetween 23 and 27 amino acid residues in length. The peptide may consistessentially of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or residues 210-240 (SEQ ID NO: 3). The peptide may compriseresidues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ IDNO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or 210-240(SEQ ID NO: 3). The peptide may consist of residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3).

In another embodiment, there is provided a nucleic acid encoding apeptide segment consisting of between 12 and 100 amino acid residues andcomprising residues 210-221, 220-236, 210-233, 210-236, 214-236 or214-240 of SEQ ID NO:1. The nucleic acid may encode a peptide of 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 70,75, 80, 85, 90, 95 or 100 amino acid residues in length. The nucleicacid may encode a peptide of between 12 and 50 amino acid residues inlength. The peptide may be between 23 and 50 amino acid residues inlength. The nucleic acid may encode a peptide of between 16 and 30 aminoacid residues in length. The nucleic acid may encode a peptide ofbetween 23 and 27 amino acid residues in length. The nucleic acid mayencode a peptide consisting essentially of residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or residues 210-240 (SEQ ID NO: 3). Thenucleic acid may encode a peptide comprising residues 210-221 (SEQ IDNO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ IDNO: 7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3). The nucleicacid may encode a peptide consisting of residues 210-221 (SEQ ID NO: 8),210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7)or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3).

In yet another embodiment, there is provided a method of inhibiting α4β1integrin (VLA-4) interaction with VEGFR2 comprising contacting a VEGFR2molecule with a peptide segment consisting of between 12 and 100 aminoacid residues and comprising 210-221, 220-236, 210-233, 210-236, 214-236or 214-240 of SEQ ID NO:1. The peptide or polypeptide may be 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 35,40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 12 and 50 amino acidresidues in length. The peptide may be between 23 and 50 amino acidresidues in length. The peptide may be between 16 and 30 amino acidresidues in length. The peptide may be between 23 and 27 amino acidresidues in length. The peptide may consist essentially of residues210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2),214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or residues 210-240(SEQ ID NO: 3). The peptide may comprise residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3). The peptide mayconsist of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or 210-240 (SEQ ID NO: 3). The α4β1 integrin (VLA-4) and/or VEGFR2may be located on the surface of a cell, such as a lymphoid cell,including a plasma cell, or a vascular endothelial cell or a lymphaticendothelial cell. The cell may also be a cancer cell, such as acarcinoma, a myeloma (including multiple myeloma), a leukemia (includingCLL), a lymphoma, a melanoma, a schwannoma, a malignant peripheral nervesheath tumor cell, a malignant endothelial cell or a glioma. The methodmay further comprise contacting said cancer cell with a second cancerinhibitory agent. The cancer cell may be a metastatic cancer cell ortumor stem cell. The step of contacting may comprise providing to saidcell an expression construct comprising a nucleic acid encoding apeptide segment consisting of between 12 and 100 amino acid residues andcomprising residues 210-221, 210-233, 210-236 or 214-236 or 214-240 ofSEQ ID NO:1 operably linked to a promoter active in said cell.

In yet another embodiment, there is provided a method of inhibiting α4β1integrin (VLA-4) interaction with syndecan-1 comprising contacting asyndecan-1 molecule with a peptide segment consisting of between 12 and100 amino acid residues and comprising 210-221, 210-233, 210-236, or210-240 of SEQ ID NO:1. The peptide or polypeptide may be 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 35,40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 12 and 50 amino acidresidues in length. The peptide may be between 14 and 50 amino acidresidues, 23 and 50 amino acid residues in length. The peptide may bebetween 16 and 30 amino acid residues in length. The peptide may bebetween 23 and 27 amino acid residues in length. The peptide may consistessentially of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), or residues 210-240 (SEQ ID NO: 3). The peptidemay comprise residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), or 210-240 (SEQ ID NO: 3). The peptide mayconsist of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), or 210-240 (SEQ ID NO: 3). The α4β1 integrin(VLA-4) and/or syndecan-1 may be located on the surface of a cell, suchas a lymphoid cell, including a plasma cell, or a vascular endothelialcell or a lymphatic endothelial cell. The cell may also be a cancercell, such as a carcinoma, a myeloma (including multiple myeloma), aleukemia (including CLL), a lymphoma, a melanoma, a schwannoma, amalignant peripheral nerve sheath tumor cell, a malignant endothelialcell or a glioma. The method may further comprise contacting said cancercell with a second cancer inhibitory agent. The cancer cell may be ametastatic cancer cell or tumor stem cell. The step of contacting maycomprise providing to said cell an expression construct comprising anucleic acid encoding a peptide segment consisting of between 24 and 100amino acid residues and comprising residues 210-221, 210-233, 210-236 or210-240 of SEQ ID NO: 1 operably linked to a promoter active in saidcell.

In still yet a further embodiment, there is provided a method ofscreening for an agent that inhibits the binding of syndecan-1 andeither VLA-4 or VEGFR2 comprising (a) providing a syndecan-1 or afragment thereof and VLA-4 or VEGFR2, or fragments thereof, wherein saidsyndecan-1 or a fragment thereof and VLA-4 or VEGFR2, or fragmentsthereof are capable of binding each other; (b) contacting the proteinsor fragments of step (a) with a candidate substance; and (c) assessingthe binding of said syndecan-1 or a fragment thereof and VLA-4 orVEGFR2, or fragments thereof, wherein reduced binding in step (c) ascompared to the binding in the absence of said candidate substanceidentifies said candidate substance as an agent that inhibits thebinding of syndecan-1 and VLA-4 or VEGFR2, or fragments thereof. Thecandidate substance may be a protein, a peptide, a peptidometic, apolynucleotide, an oligonucleotide, or a small molecule. One or both ofsaid syndecan-1 or a fragment thereof and VLA-4 or VEGFR2, or fragmentsthereof may be labeled with a detectable label. Step (c) may compriseFRET, immunodetection, a gel-shift assay, or a phosphorylation assay.The candidate substance may be a peptide segment consisting of between20 and 100 amino acid residues and comprising residues 210-221, 210-233,210-236, 214-236 or 214-240 of SEQ ID NO: 1. Step (a) may furthercomprise including VLA-4 or a fragment thereof that interacts withsyndecan-1 and/or VLA-4 and/or VEGFR2, or fragments thereof. The methodmay further comprise a control reaction of assessing the binding of saidsyndecan-1 or a fragment thereof and said VEGFR2 or a fragment thereofin the absence of said candidate substance. Steps (a)-(c) may beperformed in a cell-free system, performed in a cell or performed invivo.

In a further embodiment, there is provided a method of treating asubject with a cancer, cancer cells of which express VLA-4 and/orVEGFR2, comprising contacting said cells with a peptide segmentconsisting of between 12 and 100 amino acid residues and comprisingresidues 210-221, 220-236, 210-233, 210-236, 214-236 or 214-240 of SEQID NO: 1. The peptide may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 12 and 50 amino acidresidues in length. The peptide may be between 23 and 50 amino acidresidues in length. The peptide may be between 16 and 30 amino acidresidues in length. The peptide may be between 23 and 27 amino acidresidues in length. The peptide may consist essentially of residues210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2),214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or residues 210-240(SEQ ID NO: 3). The peptide may comprise residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3). The peptide mayconsist of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or 210-240 (SEQ ID NO: 3). The subject may be a human or a non-humanmammal. The cancer may be a carcinoma, a leukemia (including CLL), alymphoma, a myeloma (including multiple myeloma), a melanoma or aglioma. The peptide may be administered directly to said cancer cells,local to said cancer cells, regional to said cancer cells, orsystemically. The method may further comprise administering to saidsubject a second cancer therapy selected from chemotherapy,radiotherapy, immunotherapy, hormonal therapy, or gene therapy. Themethod may further comprise administering said peptide to said subjectmore than once.

In yet a further embodiment, there is provided a method of inhibitingpathologic scarring or wound repair in a subject to comprisingadministering to said subject a peptide segment consisting of between 12and 100 amino acid residues and comprising resides 210-221, 220-236,210-233, 210-236, 214-236 or 214-240 of SEQ ID NO:1. The peptide orpolypeptide may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 12 and 50 amino acidresidues in length. The peptide may be between 23 and 50 amino acidresidues in length. The peptide may be between 16 and 30 amino acidresidues in length. The peptide may be between 23 and 27 amino acidresidues in length. The peptide may consist essentially of residues210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2),214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or residues 210-240(SEQ ID NO: 3). The peptide may comprise residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3). The peptide mayconsist of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or 210-240 (SEQ ID NO: 3).

In still yet a further embodiment, there is provided a method ofinhibiting pathologic neovascularization or lymphangiogenesis comprisingadministering to said subject a peptide segment consisting of between 12and 100 amino acid residues and comprising residues 210-221, 220-236,210-233, 236, 214-236 or 214-240 of SEQ ID NO: 1. The peptide orpolypeptide may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may be between 16 and 50 amino acidresidues in length. The peptide may be between 12 and 50 amino acidresidues in length. The peptide may be between 16 and 30 amino acidresidues in length. The peptide may be between 23 and 27 amino acidresidues in length. The peptide may consist essentially of residues210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2),214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or residues 210-240(SEQ ID NO: 3). The peptide may comprise residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3). The peptide mayconsist of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or 210-240 (SEQ ID NO: 3). The pathological neovascularization mayinvolve activated vascular endothelial cells.

In an additional embodiment, there is provided a method of promotingwound healing comprising contacting an injured tissue site withsyndecan-1 or an active fragment thereof. The injured tissue site may bea surgery site, a trauma site, or a site of hypoxic injury. The site ofhypoxic injury is infarcted myocardium. The syndecan-1 fragment maycomprise residues 210-236 (SEQ ID NO: 2) or 210-240 (SEQ ID NO: 3).

Another embodiment comprises a method of treating an inflammatory orautoimmune disease or immune rejection of transplanted organs comprisingadministering to a subject a peptide segment consisting of between 12and 100 amino acid residues and comprising resides 210-221, 220-236,210-233, 210-236, 214-236 or 214-240 of SEQ ID NO: 1. The disease may berheumatoid arthritics, inflammatory bowel disease, Crohn's disease,multiple sclerosis, or systemic lupus erythematosus. Administering maycomprise systemic administration or administration local or regional toan affected disease site. The peptide or polypeptide may be 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 70, 75,80, 85, 90, 95 or 100 amino acid residues in length. The peptide may bebetween 16 and 50 amino acid residues in length. The peptide may bebetween 12 and 50 amino acid residues in length. The peptide may bebetween 16 and 30 amino acid residues in length. The peptide may bebetween 23 and 27 amino acid residues in length. The peptide may consistessentially of residues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4),210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO:5), or residues 210-240 (SEQ ID NO: 3). The peptide may compriseresidues 210-221 (SEQ ID NO: 8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ IDNO: 2), 214-236 (SEQ ID NO: 7) or 214-240 (SEQ ID NO: 5), or 210-240(SEQ ID NO: 3). The peptide may consist of residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO: 4), 210-236 (SEQ ID NO: 2), 214-236 (SEQ ID NO:7) or 214-240 (SEQ ID NO: 5), or 210-240 (SEQ ID NO: 3).

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Consisting essentially thereof, as used herein, means that the peptidecontains other elements that do not substantially alter the function ofthe peptide without that element.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed.

FIGS. 1A-D. Adhesion of CAG cells to FN or VCAM-1 by α4β1-integrin isenhanced by HPSE leading to cell spreading and migration. (FIG. 1A)HPSE^(low) or HPSE^(high) cells were plated on FN or VCAM-1 for 2.5 hrwith or without treatment of HPSE inhibitor SST0001 or 10 μg/ml α4blocking antibody (clone P1H4) and stained with fluorescent phalloidin.CAG cells expressing low or high levels of HPSE were treated with 500μg/ml of the HPSE inhibitor SST0001 for 24 hr prior to plating. (FIG.1B) Quantification of HPSE^(low) and HPSE^(high) cells attachment andspreading on 40 μg/ml FN or 5 μg/ml VCAM-1 in the presence or absence ofHPSE inhibitor SST0001 and 10 μg/ml α4 blocking antibody. (FIG. 1C)HPSE^(low) and HPSE^(high) cells were pretreated with heparinase III for2.5 hrs and then plated on FN and VCAM-1. (FIG. 1D) Transwell migrationassays towards FN (for 12 hrs) were performed with HPSE^(low) andHPSE^(high) cells. Cell migrating to the bottom-side of the filter werecounted in five random images for each experiment. Error barsrepresented S.D.

FIGS. 2A-C. MMP-9-mediated shedding of Sdc1 is necessary for theHPSE-enhanced effect in CAG cells. (FIG. 2A) HPSE^(low) and HPSE^(high)cells were plated on VCAM-1 for 2.5 hrs in the absence or presence of 10μg/ml MMP9 blocking antibody and stained with fluorescent phalloidin.(FIG. 2B) HPSE^(low) and HPSE^(high) cells were seeded at equal densityand grown in serum-free media for 2.5 hr in the absence or presence of20 μg/ml MMP9 blocking antibody. After 2.5 hr, conditioned media wereharvested, and the level of shed Sdc1 in the conditioned medium fromeach condition was determined by immunoblotting following heparinasedigestion. (FIG. 2C) HPSE^(high) cells were plated on FN in the absenceor presence of 10 μg/ml MMP blocking antibody with or without treatmentwith GST-S1ED.

FIGS. 3A-C. Amino acids 210-236 in the Sdc1 ectodomain can mimic theeffects of HPSE. (FIG. 3A) HPSE^(low) cells were plated on VCAM-1 andtreated with 4 μM GST-S1ED constructs shown (Bar=50 μm); (FIG. 3B)Schematic presentation of GST-tagged S1ED constructs and S1ED peptidesused and a summary of their ability to induce spreading indicative ofthe invasive phenotype; (FIG. 3C) HPSE^(low) cells were plated on VCAM-1in the absence or presence of 0.3 μM S1ED²¹⁰⁻²⁴⁰, S1ED²¹⁰⁻²³⁶,S1ED²¹⁰⁻²³³ or S1ED²¹⁴⁻²⁴⁰ peptide. (Bar=50 μm). Sequences in FIG. 3Care 210-240 (SEQ ID NO: 3), 210-236 (SEQ ID NO: 2), 210-233 (SEQ ID NO:4) and 214-240 (SEQ ID NO: 5).

FIGS. 4A-C. HPSE expression leads to activation of VEGFR2 whenα4-integrin engages ligand. (FIG. 4A) HPSE^(low) and HPSE^(high) cellstreated with or without 1 μM Vandetanib, 20 ng/ml VEGF, or 20 μg/mlVEGFR2 blocking antibody are plated on VCAM-1 to observe cell spreading(Bar=50 μm); (FIG. 4B) HPSE^(high) cells plated on FN were treated withMMP-9 blocking antibody or 1 μM Vandetanib in the absence or presence of0.3 μM S1ED²¹⁰⁻²³⁶ (Bar=50 μm); (FIG. 4C) HPSE^(low) cells were kept insuspension or were seeded on FN in the absence or presence of 0.3 μMS1ED²¹⁰⁻²³⁶ for 2.5 h. Cell lysates were immunoblotted with antibodiesagainst pY1054/1059 in VEGFR2 as a marker of VEGFR2 activation. Actin isshown as a loading control.

FIGS. 5A-C. Recombinant Sdc1 ectodomain (GST-S1ED) causes capture ofVEGFR2 by VLA-4. (FIG. 5A) HPSE^(high) cells were plated on FN for 2.5hrs and double stained for VLA-4 and VEGFR2. (FIG. 5B) HPSE^(low) cellswere plated on FN in the presence of 4 μM GST-S1ED. After 2.5 hrs, cellswere double-stained for GST to detect the GST-S1ED and VLA-4 or VEGFR2.(FIG. 5C) HPSE^(low) cells were plated on FN-pre-coated dishes for 2.5hr in the absence or presence of GST-S1ED and whole cell lysates wereprepared for immunoprecipitation with anti-VEGFR2 and VLA-4 antibodies.The associated VLA-4 was detected by immunoblotting with a VLA-4antibody.

FIGS. 6A-G. Peptides derived from the active site in Sdc1 activate orinhibit adhesion and invasion. (FIG. 6A) Transwell migration assaystowards FN (for 16 hrs) were performed for HPSE low or high cells in thepresence of 0, 0.3, 3, and 30 μM of S1ED²¹⁰⁻²⁴⁰. Cells migrating to thebottom side of the filter were counted from five random images for eachtreatment. (FIG. 6B) The sequence of S1ED²¹⁰⁻²⁴⁰ is shown (SEQ ID NO:3), as well as three truncation mutants of the peptide (SEQ ID NOs: 5,2, 4 and 7). Amino acids shown in red appear necessary for the spreadingand invasive activity. (FIG. 6C) Images of HPSE^(low) and HPSE^(high)cells plated on FN for 2 hr. (FIG. 6D) HPSE^(low) cells were treatedwith 0, 0.3, 3, 10 and 30 μM of S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, orS1ED²¹⁴⁻²⁴⁰, plated on FN for 2 hr, fixed and stained with fluorescentphalloidin. Cells from five random images were quantified for cellattachment and cell spreading. (FIG. 6E) HPSE^(high) cells were treatedwith 0, 3, 10, and 30 μM of S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, and S1ED²¹⁴⁻²⁴⁰,plated on FN for 2 hr, then fixed, imaged and quantified as in FIG. 6D.(FIG. 6F) Transwell migration assays towards FN (for 16 hr) wereperformed for HPSE low or HPSE^(high) cells in the presence of 30 μM ofS1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, S1ED²¹⁴⁻²⁴⁰ and Vandetanib. (FIG. 6G)HPSE^(low) cell lysates were incubated overnight with glutathione beadscoated with GST or GST-S1ED in the absence or presence of 30 μM ofS1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, and S1ED²¹⁴⁻²⁴⁰ Capture of VLA-4 or VEGFR2 wasdetected by immunoblotting. Sequences in FIG. 6B are 210-240 (SEQ ID NO:3), 210-236 (SEQ ID NO: 2), 210-233 (SEQ ID NO: 4), 214-240 (SEQ ID NO:5) and 214-236 (SEQ ID NO: 7).

FIG. 7A-B. S1ED²¹⁴⁻²³⁶, specific for VEGFR2 coupling to VLA-4, blocksmyeloma cell invasion but not adhesion. (FIG. 7A) P3×63Ag8 mouse myelomacells are plating on the VLA-4 ligand FN in the presence or absence of30 uM S1ED²¹⁰⁻²³³ (SEQ ID No: 4) or S1ED²¹⁴⁻²³⁶ (SEQ ID No: 7) for 2.5hr. (FIG. 7B) P3×63Ag8 myeloma cells are plated on FN-coated filters inthe presence of HPSE inhibitor OGT2115, VLA-4 blocking antibody P1H4,VEGFR2 inhibitor Vandetanib or S1ED²¹⁰⁻²³³ or S1ED²¹⁴⁻²³⁶ and allowed tomigrate through the filter for 16 hr. Small black dots are the pores inthe filter. Cell migration is only observed in the untreated controls.

FIGS. 8A-H. HPSE mediated the Sdc1-coupled VEGFR2 complex in endothelialcells induces angiogenesis. (FIG. 8A) Lysates from HPSE^(low),HPSE^(high) and HMEC-1 cells are probed by immunoblotting for expressionof HPSE. (FIG. 8B) HMEC-1 cells were seeded at equal density and grownin serum-free media for 24 hr in the absence or presence of 125 μg/ml ofthe HPSE inhibitor SST0001. After 24 hr, conditioned media wereharvested, and the level of shed Sdc1 in the conditioned medium fromeach condition was determined by immunoblotting. (FIG. 8C) HMEC-1 celllysate was incubated overnight with glutathione beads coated with GST orGST-S1ED. Capture of VLA-4 or VEGFR2 was detected by immunoblotting.(FIG. 8D) HMEC-1 cells were plated on IIICS for 3 hr and double stainedfor VLA-4 and Sdc1, VEGFR2 and Sdc1, or VLA-4 and VEGFR2. (FIG. 8E)HMEC-1 cells were treated with DMSO, VLA-4 blocking antibody P1H4,β1-integrin blocking antibody mAb13, g/ml MMP9 blocking antibody, 125μg/ml HPSE inhibitor SST0001, Vandetanib, VEGFR2 blocking antibody, 30μM S1ED²¹⁰⁻²³³ or 30 μM S1ED²¹⁴⁻²⁴⁰ and then plated on IIICS. Imageswere taken from random field with 200× magnification for countingattached and spread cells. (FIG. 8F) HMEC-1 cells were plated on IIICSin the presence of 10 μM of S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, S1ED²¹⁵⁻²⁴⁰, andS1ED²¹⁴⁻²⁴⁰. Lysates were probed on immunoblots with antibodies againstpY¹¹⁷⁵ VEGFR2 and VEGFR2. (FIG. 8G) Transwell migration assays towardsIIICS (for 16 hr) were performed for HMEC-1 cells in the presence ofP1H4, SST0001, Anti-MMP9, Vandetanib, S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³, andS1ED²¹⁴⁻²⁴⁰. Images were taken from random field with 200× magnificationfor counting migrated cells. (FIG. 8H) A 96-well plate coated with 50 μlMatrigel containing 100 μg/ml IIICS per well was solidified at 37° C.for 2 hrs. HMEC-1 cells (2.5×10⁴ cells/well) were seeded into the plateand cultured in media containing VEGF, SST0001, 10 μM of S1ED²¹⁰⁻²³⁶,S1ED²¹⁰⁻²³³, S1ED²¹⁵⁻²⁴⁰, and S1ED²¹⁴⁻²⁴⁰ for 24 hrs. Enclosed capillarynetworks of tubes were taken from random field with 200× magnification.

FIG. 9. Treatment of breast cancer xenograft with systemic S1ED²¹⁰⁻²⁴⁰.4×10⁶ human SKBr3 mammary carcinoma cells were injected in a 1:1 mixwith matrigel into the posterior flanks of immunodeficient nude (nu/nu)mice and allowed to establish for one week as palpable tumors. Alzetpumps were then implanted subcutaneously on the anterior dorsal backs ofthe animals, systemically delivering either phosphate buffered-salinealone (PBS) or S1ED 210-240 peptide in PBS for 3 weeks of treatment.Peptide was delivered at 1.09 mg/kg/day, a concentration estimated toachieve concentrations sufficient to block the VLA-4/VEGFR2 mechanism.Tumor sizes were measured with calipers and converted to tumor volumesusing the equation V=0.524× L× W. The data are shown as the mean of 12tumors+/−S.E.M.

FIG. 10. Human Syndecan-1 Sequences. Full length syndecan-1 sequencesand relevant fragments are illustrated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, receptor tyrosine kinases and integrin play a majorrole in oncogenesis, often working together. A significant question thathas remained unanswered is whether the activity of these two classes ofreceptors are jointly regulated, perhaps by a third “organizer”receptor. The inventors' laboratory has previously shown that syndecansappear to be the organizer. The focus here will be on Sdc1, which isexpressed on epithelial cells (Bernfield et al., 1992; David et al.,1992), but also endothelial and myeloma cells (Beauvais et al., 2009;Sanderson and Bernfield, 1988; Liu et al., 1998). Previously, thismolecule was shown to co-immunoprecipitate with the αvβ3 integrin andIGF-1R from breast carcinoma and activated endothelial cells (Beauvaiset al., 2009; Beauvais et al., 2010). It captures the integrin andreceptor tyrosine kinase via a site in its extracellular domain (aminoacids 93-120 in human Sdc1) that is unique to this syndecan. Activationof the integrin and signaling by IGF-1R requires their capture by Sdc1,which the inventors report is disrupted by a peptide mimetic (called a“synstatin” or SSTN) of the active site (e.g., SSTN_(IGF1R)). Morerecently it has been shown that Sdc1 also co-immunoprecipitates with theα6β4 integrin, α3β1 integrin and HER2 from activated keratinocytes, A431cervical carcinoma cells, breast carcinoma cells and HN squamouscarcinoma cells (Wang et al., 2010; Wang et al., 2014; Wang et al.,2015). Whereas the 6034 integrin is captured by the syndecan cytoplasmicdomain, HER2 and the α3β1 are captured by a site in the extracellulardomain, that is distinct from the αvβ3/IGF-1R capture site. Importantly,HER2 docks only with Sdc1/α3β1 and not with the other syndecans. Theinteraction site capturing HER2/α3β1 is distinct from other syndecans,and the inventors' laboratory has reported that recombinant Sdc1peptides mimicking this interaction site (designated as “synstatin-HER2”or “SSTN_(HER2)”) block this interaction. Sdc1 binding its site in theβ4 integrin cytoplasmic domain is essential for signaling by thiscomplex, as mutation of this Sdc1-specific site generates a β4 dominantnegative mutant (DNM) that specifically blocks the Sdc1-coupledsignaling mechanism.

In the work described here, the inventors examine the role of HPSE onmyeloma cell adhesion and a potential link to Sdc1 acting as anorganizer of matrix- and growth factor-dependent signaling. They nowshow that human CAG myeloma cells bind via α4-integrin (also called“very late antigen-4” or VLA-4) to fibronectin (FN), an abundant bonemarrow matrix ligand, and to vascular cell adhesion molecule-1 (VCAM-1),a receptor found on microvascular endothelial cells and on stromal cellswithin the bone marrow microenvironment^(3, 4). CAG cells expressingelevated levels of HPSE adopt an invasive phenotype, characterized bypolarized spreading and directional cell migration on these ligands. TheHPSE-induced phenotype depends on the shedding of Sdc1, which can bemimicked by addition of soluble recombinant Sdc1 ectodomain or a peptide(a.a. 210-236) that represents a novel active site in the syndecanextracellular domain. The phenotype also depends on activation ofVEGFR2, although activation of VEGFR2 by its ligand, VEGF, rather thanby Sdc1 does not cause the phenotype. Instead, Sdc1 couples VEGFR2 toVLA-4, which, when engaged and clustered by FN or VCAM-1, activatesVEGFR2 and stimulates the invasive phenotype. Low concentrations ofS1ED²¹⁰⁻²³⁶ stimulate the invasive phenotype, whereas higherconcentration block the phenotype in CAG cells expressing high levels ofHPSE. This behavior is typical of a peptide that has binding sites fortwo receptors, such that at low concentrations a single peptide bridgesboth receptors and couples them together, whereas at higherconcentrations single peptides occupy the binding sites on individualreceptors and prevent any single peptide from bridging and coupling themtogether. Analysis of binding sites within this peptide reveal thatVLA-4 requires a binding site at the N-terminus (amino acids 210-213),whereas VEGFR2 requires amino acids near the C-terminus (amino acids234-236). Peptides bearing only one of these binding sites bind only asingle receptor and prevent endogenous Sdc1 from coupling the receptorstogether, thus acting as inhibitors. The peptide that engages only VLA-4inactivates the integrin, completely blocking adhesion by the cells. Thepeptide that engages only VEGFR2 does not block adhesion, but blocks theVLA-4-mediated invasion by the cells. The inventors also find thatvascular endothelial cells, which normally express VLA-4 and VEGFR2,also depend on this mechanism to respond to VLA-4 ligands duringangiogenesis. These and other aspects of the disclosure are discussedbelow.

I. SYNDECANS

A. The Syndecan Family

Cell surface adhesion receptors physically bind cells to theirextracellular matrix (ECM) and couple such interactions to intracellularsignaling mechanisms which influence gene expression, cell morphology,motility, growth, differentiation and survival (Roskelley et al., 1995;Miranti and Brugge, 2002). Many ECM ligands contain closely spacedproteoglycan- and integrin-binding domains, indicating that themolecular mechanisms by which cells recognize and interact with theirextracellular milieu may involve the formation of signaling complexescontaining both proteoglycans and integrins. Consequentially, these twotypes of receptors may act in concert to modulate cell adhesion andmigration. While the role of integrins in cell adhesion and signaling iswell established, the role of heparan sulfate proteoglycans (HSPGs) isnot well characterized.

The vertebrate syndecans are a family of four transmembrane HSPGs.Endowed by their heparan sulfate (HS) chains, syndecans bind a varietyof ECM ligands, including fibronectin (FN), laminin (LN), tenascin,thrombospondin (TSP), vitronectin (VN) and the fibrillar collagens (COL)(Bernfield et al., 1999). While the syndecan HS chains are essential formatrix binding, less is known about the role of syndecan core proteinsin cell adhesion signaling, although the core protein can affect ligandbinding interactions, as well as occupancy induced signaling (Rapraegerand Ott, 1998; Rapraeger, 2000).

The syndecans display a high degree of conservation within their coreproteins both across species and across family members. Like theintegrins, the syndecans lack intrinsic signaling activity. Their shortcytoplasmic tails (ca. 30 a.a.) consist of three regions, two of whichare conserved amongst the four syndecans (C1 and C2) and which flank anintervening variable (V) region. Proteins known to interact with theseconserved domains are believed to link syndecan ligand bindinginteractions to the transduction of intracellular signals (Couchman etal., 2001). Each family member is uniquely defined by its ectodomainsand the V-regions of its cytoplasmic tail. Divergence within theseregions is believed to confer separate and distinct functions to eachindividual family member. Distinct roles for the V-regions of Sdc-2 and-4 in matrix assembly and focal adhesion formation respectively havebeen described (Klass et al., 2000; Woods and Couchman, 2001); however,specific functions for the syndecan ectodomains are almost whollyunknown with the noted exception of the inventors' work on Sdc-1, whichcontains binding site for αvβ3 integrin/IGF-1R or α6β4 integrin/HER2 andSdc-4 in which the inventors describe a binding site for as yetunidentified cell surface receptor(s) (McFall and Rapraeger, 1997;McFall and Rapraeger, 1998) and α6β4 integrin/EGFR (Wang et al.,unpublished).

B. Syndecan Function in Cell Adhesion and Spreading

Current evidence suggests that the syndecan core proteins participate inadhesion-mediated signaling in collaboration with co-receptors at thecell surface. One example is Sdc-4 in focal adhesion and stress fiberformation, which requires both Sdc-4 and integrin engagement whereasneither is sufficient alone (Woods et al., 1986; Izzard et al., 1986;Streeter and Rees, 1987; Singer et al., 1987). The requirement for Sdc-4ligation can be overcome by treatment with phorbol esters (Woods andCouchman, 1994) or lysophosphatidic acid (LPA) (Saoncella et al., 1999)implicating PKC and RhoA in Sdc-4 signaling. While the mechanism bywhich Sdc-4 contributes to RhoA activation is not clear, it is knownthat Sdc-4 interacts directly with PKCa as well as phosphatidyl inositol4,5 bisphosphate (PIP2) via its cytoplasmic tail and these interactionspotentiate PKCa activity (Oh et al., 1997a; Oh et al., 1997b; Oh et al.,1998; Baciu and Goetinck, 1995).

While the mechanism by which Sdc-1 signals is not clear, there is ampleevidence implicating a signaling role for this receptor as well. Ectopicexpression of Sdc-1 in Schwann cells enhances cell spreading andpromotes the formation of focal adhesions (Hansen et al., 1994) andactin stress fibers (Carey et al., 1994a); similar morphological changesoccur when Sdc-1 is co-clustered with antibodies (Carey et al., 1994b).This response requires the cytoplasmic domain, since clustering of atruncated core protein did not induce reorganization of thecytoskeleton. Expression of Sdc-1 in human ARH-77 leukemia cells orhepatocellular carcinoma cells inhibits invasion of cells into COLmatrices (Liu et al., 1998; Ohtake et al., 1999). ARH-77 cellsexpressing a chimera comprised of the Sdc-1 ectodomain fused to theglycosyl-phosphatidyl inositol (GPI) tail of glypican-1 also fail toinvade a COL matrix demonstrating that Sdc-1's anti-invasive activityresides in its extracellular domain. In similar studies, Raji humanlymphoblastoid cells transfected with mouse Sdc-1 (Raji-S1) spread onTSP, FN and antibodies directed against the Sdc-1 ectodomain (Lebakkenand Rapraeger, 1996). This spreading is unaffected by truncation of thecytoplasmic domain, indicating that the Sdc-1 core protein interactswith and cooperatively signals through an associated transmembranesignaling partner. Analogous features have also been observed for Sdc-2(Granes et al., 1999) and Sdc-4 (Yamashita et al., 1999).

Potential syndecan signaling partners include cell surface integrins.Iba et al. (2000) demonstrated that mesenchymal cells when seeded on anHS-specific ligand, the cysteine rich domain of a disintegrin andmetalloprotease, ADAM-12/Meltrin α (rADAM12-cys), will spread in amanner that requires cooperate signaling of both syndecans and β₁integrins. These results imply that syndecan(s) can trigger signalingcascades required for cell spreading either by exposing a crypticbinding site for β₁ integrins, as has been proposed for FN (Khan et al.,1988), or by modulating the activation state of β₁ integrins.Interestingly, colon carcinoma cells attach but fail to spread onaADAM12-cys. However, exogenous stimulation of β₁ integrins with Mn²⁺ orβ₁ integrin function activating antibody, mAb 12G10, induced cellspreading, suggesting a mechanism whereby the syndecan activates β₁integrins is blocked in transformed cells.

C. Syndecan-1

Syndecan-1 is highly expressed at the basolateral surface of epithelialcells where it is thought to interact with the actin cytoskeleton and tomodulate cell adhesion and growth factor signaling (Bernfield et al.,1999; Rapraeger et al., 1986; Kim et al., 1994; Sanderson and Bernfield,1988). In experimental studies of malignant transformation, Sdc-1expression is associated with the maintenance of epithelial morphology,anchorage-dependent growth and inhibition of invasiveness. Alterationsin syndecan expression during development (Sun et al., 1998) and intransformed epithelial (Inki and Jalkanen, 1996; Bayer-Garner et al.,2001) are associated with an epithelial-mesenchymal transformation withattendant alterations in cell morphology, motility, growth anddifferentiation. Transfection of epithelial cells with anti-sense mRNAfor Sdc-1 or downregulation of Sdc-1 expression by androgen-inducedtransformation results in an epithelial to mesenchymal transformationand increased invasion (Leppa et al., 1992; Kato et al., 1995; Leppa etal., 1991). The loss of E-cadherin under these circumstances has longsuggested a coordinate regulation of Sdc-1 and E-cadherin expression(Sun et al., 1998; Leppa et al., 1996). These studies, as well asothers, indicate that there appears to be a threshold requirement forsyndecan expression to elicit its biological activity. Syndecan-1 isdownregulated in a number of epithelial cancers and in pre-malignantlesions of the oral mucosa (Soukka et al., 2000) and uterine cervix(Inki et al., 1994; Rintala et al., 1999; Nakanishi et al., 1999), andits loss may be an early genetic event contributing to tumor progression(Sanderson, 2001; Numa et al., 2002; Hirabayashi et al., 1998). Loss ofSdc-1 correlates with a reduced survival in squamous cell carcinoma ofthe head, neck and lung (Anttonen et al., 1999; Inki et al., 1994;Nakaerts et al., 1997), laryngeal cancer (Pulkkinen et al., 1997;Klatka, 2002), malignant mesothelioma (Kumar-Singh et al., 1998) andmultiple myeloma (Sanderson and Borset, 2002) and a high metastaticpotential in hepatocellular and colorectal carcinomas (Matsumoto et al.,1997; Fujiya et al., 2001; Levy et al., 1997; Levy et al., 1996).Downregulation of Sdc-2 and -4 expression has also been observed incertain human carcinomas (Nakaerts et al., 1997; Park et al., 2002;Mundhenke et al., 2002; Crescimanno et al., 1999), but the functionalconsequences of these alterations in expression are less clear.

In contrast to the general notion that the syndecan may be an inhibitorof carcinogenesis, Sdc-1 also demonstrates tumor promoter function.Syndecan-1 supplements Wnt-1 induced tumorigenesis of the mouse mammarygland (Alexander et al., 2000) and promotes the formation of metastasesin mouse lung squamous carcinoma cells (Hirabayashi et al., 1998).Enhanced Sdc-1 expression has also been observed in pancreatic (Conejoet al., 2000), gastric (Wiksten et al., 2001) and breast (Burbach etal., 2003; Stanley et al., 1999; Barbareschi et al., 2003) carcinomasand this overexpression correlates with increased tumor aggressivenessand poor clinical prognosis. This duality in the role of Sdc-1 intumorigenesis may reflect tissue and/or tumor stage-specific function,or reflect the multiple functions of this PG.

Sanderson was the first to demonstrate a role for Sdc-1 in tumor cellmigration by examining the invasion of myeloma cells into collagen gels(Liu et al., 1998). Ectopic expression of Sdc-1 in syndecan-deficientmyeloma cells had the striking effect of curtailing invasion, whereasthe expression of other cell surface heparan sulfate PGs (e.g.,glypican) was without effect. Using chimeras derived from these twoproteins, Sanderson showed that the activity of the syndecan ispreserved when its ectodomain alone is expressed as aglycosyl-phosphatidylinositol (GPI)-linked protein at the cell surface.Although clearly responsible for binding the collagen matrix via itsattached heparan sulfate chains, the anti-invasive activity of thesyndecan requires yet an additional interaction that traces to a site inthe extracellular domain of the core protein itself. The mechanism bywhich the ectodomain site influences the invasion of the myeloma cellsis unknown, but its interaction with other cell surface receptors in a“co-receptor” role is one possibility. More recently, ectopic expressionof Sdc-1 has also been shown to curtail the invasion of hepatocellularcarcinoma cells into a collagen matrix (Ohtake et al., 1999).

II. INTEGRINS AND VEGFR2

A. VLA-4 (α4β1 Integrin)

Integrins are receptors that mediate attachment between a cell and thetissues surrounding it, which may be other cells or the ECM. They alsoplay a role in cell signaling and thereby regulate cellular shape,motility, and the cell cycle.

Typically, receptors inform a cell of the molecules in its environmentand the cell responds. Not only do integrins perform this outside-insignalling, but they also operate an inside-out mode. Thus, theytransduce information from the ECM to the cell as well as reveal thestatus of the cell to the outside, allowing rapid and flexible responsesto changes in the environment, for example to allow blood coagulation byplatelets.

There are many types of integrin, and many cells have multiple types ontheir surface. Integrins are of vital importance to all animals and havebeen found in all animals investigated, from sponges to mammals.Integrins have been extensively studied in humans.

Integrins work alongside other proteins such as cadherins,immunoglobulin superfamily cell adhesion molecules, selectins andsyndecans to mediate cell-cell and cell-matrix interaction andcommunication. Integrins bind cell surface and ECM components such asfibronectin, vitronectin, collagen, and laminin.

Integrin α4β1 (Very Late Antigen-4) is an integrin dimer. It is composedof CD49d (α4) and CD29 (β1). Vascular cell adhesion molecule-1(VCAM-1—an integrin receptor) located on an endothelial cell, binds toVLA-4 which are normally expressed on leukocyte plasma membranes, butthey do not adhere to their appropriate ligands until the leukocytes areactivated by chemotactic agents or other stimuli (often produced by theendothelium or other cells at the site of injury). Only then do theintegrins undergo the conformational change necessary to confer highbinding affinity for the endothelial adhesion molecules.

In multiple sclerosis, the VLA-4 integrin is essential in the processesby which T-cells gain access to the brain by allowing the cells topenetrate the blood brain barrier that normally restricts immune cellaccess. One approach to prevent an autoimmune reaction has been to blockthe action of VLA-4 so that self-reactive T-cells are unable to enterthe brain and thus unable to attack myelin protein.

Vascular endothelial and lymphatic endothelial cells also depend onVLA-4. VLA4 expression is required for angiogenesis by both vascular andlymphatic endothelial cells and its activity is especially prominent intumors. Its matrix ligand, FN, is deposited within the growing vascularand lymphatic microvessels and VCAM-1 is prominently expressed on muralcells (pericytes) that support vascular endothelial cells.¹³⁻¹⁵ Inrecent years antagonists of VLA-4 have shown great promise in treatinginflammatory disorders in a number of animal models. However, the usageof Natalizumab, an antagonist of VLA-4 integrin, remains controversialdue to several side effects including Progressive multifocalleukoencephalopathy.

B. VEGFR2

VEGF receptors are receptors for vascular endothelial growth factor(VEGF). There are three main subtypes of VEGFR, numbered 1, 2 and 3.Also, they may be membrane-bound (mbVEGFR) or soluble (sVEGFR),depending on alternative splicing.

Vascular endothelial growth factor (VEGF) is an important signalingprotein involved in both vasculogenesis (the formation of thecirculatory system) and angiogenesis (the growth of blood vessels frompre-existing vasculature). As its name implies, VEGF activity isrestricted mainly to cells of the vascular endothelium, although it doeshave effects on a limited number of other cell types (e.g., stimulationmonocyte/macrophage migration). In vitro, VEGF has been shown tostimulate endothelial cell mitogenesis and cell migration. VEGF alsoenhances microvascular permeability and is sometimes referred to asvascular permeability factor.

All members of the VEGF family stimulate cellular responses by bindingto tyrosine kinase receptors (the VEGFRs) on the cell surface, causingthem to dimerize and become activated through transphosphorylation. TheVEGF receptors have an extracellular portion consisting of 7immunoglobulin-like domains, a single transmembrane spanning region andan intracellular portion containing a split tyrosine-kinase domain.

VEGF-A binds to VEGFR1 (Flt-1) and VEGFR2 (Kinase insert domainreceptor; KDR/Flk-1). VEGFR2 appears to mediate almost all of the knowncellular responses to VEGF. The function of VEGFR1 is less well defined,although it is thought to modulate VEGFR2 signaling. Another function ofVEGFR-1 is to act as a dummy/decoy receptor, sequestering VEGF fromVEGFR2 binding (this appears to be particularly important duringvasculogenesis in the embryo). In fact, an alternatively spliced form ofVEGFR1 (sFlt1) is not a membrane bound protein but is secreted andfunctions primarily as a decoy. A third receptor has been discovered(VEGFR3), however, VEGF-A is not a ligand for this receptor. VEGFR3mediates lymphangiogenesis in response to VEGF-C and VEGF-D.

KDR (VEGFR2), a type III receptor tyrosine kinase, has also beendesignated as CD309 (cluster of differentiation 309). KDR is also knownas Flk1 (Fetal Liver Kinase 1). KDR has been shown to interact withSHC2, Annexin A5 and SHC1.

III. SYNDECAN PEPTIDES

A. Structure

The present disclosure contemplates the design, production and use ofvarious syndecan peptides. The structural features of these peptides areas follows. First, the peptides have about 23 consecutive residues of asyndecan and up to 100 consecutive residues. Thus, the term “a peptidehaving no more than X consecutive residues,” even when including theterm “comprising,” cannot be understood to comprise a greater number ofconsecutive syndecan residues. Second, the peptides will contain themotifs responsible for interaction with VEGFR2 or VLA-4. In general, thepeptides will have, at a minimum, 25 or more consecutive residues of thesyndecan.

In general, the peptides will be 100 residues or less, again, comprisingno more than 23-100 consecutive residues of a syndecan. The overalllength may be 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90or 100 residues. Ranges of peptide length of 23-50 residues, 23-27residues, 23-30, residues, 23-40 residues, 23-60, residues, 23-70residues, 23-80 residues, 23-90 residues, and 23-100 residues arecontemplated. The number of consecutive syndecan residues may be 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90 or 100 residues,including 23-50 residues, 23-27 residues, 23-30, residues, 23-40residues, 23-60, residues, 23-70 residues, 23-80 residues, 23-90residues, and 23-100.

Also as mentioned above, peptides modified for in vivo use by theaddition, at the amino- and/or carboxyl-terminal ends, of a blockingagent to facilitate survival of the peptide in vivo are contemplated.This can be useful in those situations in which the peptide termini tendto be degraded by proteases prior to cellular uptake. Such blockingagents can include, without limitation, additional related or unrelatedpeptide sequences that can be attached to the amino and/or carboxylterminal residues of the peptide to be administered. These agents can beadded either chemically during the synthesis of the peptide, or byrecombinant DNA technology by methods familiar in the art.Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino- and/orcarboxyl-terminal residues.

B. Synthesis

It will be advantageous to produce peptides using the solid-phasesynthetic techniques (Merrifield, 1963). Other peptide synthesistechniques are well known to those of skill in the art (Bodanszky etal., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia,1984). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in Protective Groups in OrganicChemistry, 1973. These synthetic methods involve the sequential additionof one or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides of the disclosure are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

Aside from the 20 standard amino acids can be used, there are a vastnumber of “non-standard” amino acids. Two of these can be specified bythe genetic code, but are rather rare in proteins. Selenocysteine isincorporated into some proteins at a UGA codon, which is normally a stopcodon. Pyrrolysine is used by some methanogenic archaea in enzymes thatthey use to produce methane. It is coded for with the codon UAG.Examples of non-standard amino acids that are not found in proteinsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine and theneurotransmitter gamma-aminobutyric acid. Non-standard amino acids oftenoccur as intermediates in the metabolic pathways for standard aminoacids—for example ornithine and citrulline occur in the urea cycle, partof amino acid catabolism. Non-standard amino acids are usually formedthrough modifications to standard amino acids. For example, homocysteineis formed through the transsulfuration pathway or by the demethylationof methionine via the intermediate metabolite S-adenosyl methionine,while hydroxyproline is made by a posttranslational modification ofproline.

C. Linkers

Linkers or cross-linking agents may be used to fuse syndecan peptides toother proteinaceous sequences. Bifunctional cross-linking reagents havebeen extensively used for a variety of purposes including preparation ofaffinity matrices, modification and stabilization of diverse structures,identification of ligand and receptor binding sites, and structuralstudies. Homobifunctional reagents that carry two identical functionalgroups proved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. In instances where a particular peptidedoes not contain a residue amenable for a given cross-linking reagent inits native sequence, conservative genetic or synthetic amino acidchanges in the primary sequence can be utilized.

Another use of linkers in the context of peptides as therapeutics is theso-called “Stapled Peptide” technology of Aileron Therapeutics. Thegeneral approach for “stapling” a peptide is that two key residueswithin the peptide are modified by attachment of linkers through theamino acid side chains. Once synthesized, the linkers are connectedthrough a catalyst, thereby creating a bridge the physically constrainsthe peptide into its native α-helical shape. In addition to helpingretain the native structure needed to interact with a target molecule,this conformation also provides stability against peptidases as well ascell-permeating properties. U.S. Pat. Nos. 7,192,713 and 7,183,059,describing this technology, are hereby incorporated by reference. Seealso Schafmeister et al. (2000).

D. Design, Variants and Analogs

Having identified structures in VEFGR2 interaction with VLA-4 integrins,the inventor also contemplates that variants of the sequences may beemployed. For example, certain non-natural amino acids that satisfy thestructural constraints of the sequences may be substituted without aloss, and perhaps with an improvement in, biological function. Inaddition, the present inventor also contemplates that structurallysimilar compounds may be formulated to mimic the key portions of peptideor polypeptides of the present disclosure. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the disclosure and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Methods for generating specific structures have been disclosed in theart. For example, α-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Methods for generatingconformationally restricted β-turns and β-bulges are described, forexample, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Othertypes of mimetic turns include reverse and γ-turns. Reverse turnmimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, andγ-turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.

As used herein, “molecular modeling” means quantitative and/orqualitative analysis of the structure and function of protein-proteinphysical interaction based on three-dimensional structural informationand protein-protein interaction models. This includes conventionalnumeric-based molecular dynamic and energy minimization models,interactive computer graphic models, modified molecular mechanicsmodels, distance geometry and other structure-based constraint models.Molecular modeling typically is performed using a computer and may befurther optimized using known methods. Computer programs that use X-raycrystallography data are particularly useful for designing suchcompounds. Programs such as RasMol, for example, can be used to generatethree dimensional models. Computer programs such as INSIGHT (Accelrys,Burlington, Mass.), GRASP (Anthony Nicholls, Columbia University), Dock(Molecular Design Institute, University of California at San Francisco),and Auto-Dock (Accelrys) allow for further manipulation and the abilityto introduce new structures. The methods can involve the additional stepof outputting to an output device a model of the 3-D structure of thecompound. In addition, the 3-D data of candidate compounds can becompared to a computer database of, for example, 3-D structures.

Compounds of the disclosure also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson, 1997;Jones et al., 1996). Candidate compounds can then be tested in standardassays familiar to those skilled in the art. Exemplary assays aredescribed herein.

The 3-D structure of biological macromolecules (e.g., proteins, nucleicacids, carbohydrates, and lipids) can be determined from data obtainedby a variety of methodologies. These methodologies, which have beenapplied most effectively to the assessment of the 3-D structure ofproteins, include: (a) x-ray crystallography; (b) nuclear magneticresonance (NMR) spectroscopy; (c) analysis of physical distanceconstraints formed between defined sites on a macromolecule, e.g.,intramolecular chemical crosslinks between residues on a protein (e.g.,PCT/US00/14667, the disclosure of which is incorporated herein byreference in its entirety), and (d) molecular modeling methods based ona knowledge of the primary structure of a protein of interest, e.g.,homology modeling techniques, threading algorithms, or ab initiostructure modeling using computer programs such as MONSSTER (Modeling OfNew Structures from Secondary and Tertiary Restraints) (see, e.g.,PCT/US99/11913, incorporated herein by reference in its entirety). Othermolecular modeling techniques may also be employed in accordance withthis disclosure (e.g., Cohen et al., 1990; Navia et al., 1992), thedisclosures of which are incorporated herein by reference in theirentirety). All these methods produce data that are amenable to computeranalysis. Other spectroscopic methods that can also be useful in themethod of the disclosure, but that do not currently provide atomic levelstructural detail about biomolecules, include circular dichroism andfluorescence and ultraviolet/visible light absorbance spectroscopy. Apreferred method of analysis is x-ray crystallography. Descriptions ofthis procedure and of NMR spectroscopy are provided below.

The present disclosure may utilize L-configuration amino acids,D-configuration amino acids, or a mixture thereof. While L-amino acidsrepresent the vast majority of amino acids found in proteins, D-aminoacids are found in some proteins produced by exotic sea-dwellingorganisms, such as cone snails. They are also abundant components of thepeptidoglycan cell walls of bacteria. D-serine may act as aneurotransmitter in the brain. The L and D convention for amino acidconfiguration refers not to the optical activity of the amino aciditself, but rather to the optical activity of the isomer ofglyceraldehyde from which that amino acid can theoretically besynthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary).

One form of an “all-D” peptide is a retro-inverso peptide. Retro-inversomodification of naturally occurring polypeptides involves the syntheticassemblage of amino acids with α-carbon stereochemistry opposite to thatof the corresponding L-amino acids, i.e., D-amino acids in reverse orderwith respect to the native peptide sequence. A retro-inverso analoguethus has reversed termini and reversed direction of peptide bonds (NH—COrather than CO—NH) while approximately maintaining the topology of theside chains as in the native peptide sequence. See U.S. Pat. No.6,261,569, incorporated herein by reference.

X-Ray Crystallography.

X-ray crystallography is based on the diffraction of x-radiation of acharacteristic wavelength by electron clouds surrounding the atomicnuclei in a crystal of a molecule or molecular complex of interest. Thetechnique uses crystals of purified biological macromolecules ormolecular complexes (but these frequently include solvent components,co-factors, substrates, or other ligands) to determine near atomicresolution of the atoms making up the particular biologicalmacromolecule. A prerequisite for solving 3-D structure by x-raycrystallography is a well-ordered crystal that will diffract x-raysstrongly. The method directs a beam of x-rays onto a regular, repeatingarray of many identical molecules so that the x-rays are diffracted fromthe array in a pattern from which the structure of an individualmolecule can be retrieved. Well-ordered crystals of, for example,globular protein molecules are large, spherical or ellipsoidal objectswith irregular surfaces. The crystals contain large channels between theindividual molecules. These channels, which normally occupy more thanone half the volume of the crystal, are filled with disordered solventmolecules, and the protein molecules are in contact with each other atonly a few small regions. This is one reason why structures of proteinsin crystals are generally the same as those of proteins in solution.

Methods of obtaining the proteins of interest are described below. Theformation of crystals is dependent on a number of different parameters,including pH, temperature, the concentration of the biologicalmacromolecule, the nature of the solvent and precipitant, as well as thepresence of added ions or ligands of the protein. Many routinecrystallization experiments may be needed to screen all these parametersfor the combinations that give a crystal suitable for x-ray diffractionanalysis. Crystallization robots can automate and speed up work ofreproducibly setting up a large number of crystallization experiments(see, e.g., U.S. Pat. No. 5,790,421, the disclosure of which isincorporated herein by reference in its entirety).

Polypeptide crystallization occurs in solutions in which the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such solutions may be restored toequilibrium by reducing the polypeptide concentration, preferablythrough precipitation of the polypeptide crystals. Often polypeptidesmay be induced to crystallize from supersaturated solutions by addingagents that alter the polypeptide surface charges or perturb theinteraction between the polypeptide and bulk water to promoteassociations that lead to crystallization.

Crystallizations are generally carried out between 4° C. and 20° C.Substances known as “precipitants” are often used to decrease thesolubility of the polypeptide in a concentrated solution by forming anenergetically unfavorable precipitating depleted layer around thepolypeptide molecules (Weber, 1991). In addition to precipitants, othermaterials are sometimes added to the polypeptide crystallizationsolution. These include buffers to adjust the pH of the solution andsalts to reduce the solubility of the polypeptide. Various precipitantsare known in the art and include the following: ethanol, 3-ethyl-2-4pentanediol, and many of the polyglycols, such as polyethylene glycol(PEG). The precipitating solutions can include, for example, 13-24% PEG4000, 5-41% ammonium sulfate, and 1.0-1.5 M sodium chloride, and a pHranging from 5.0-7.5. Other additives can include 0.1 M Hepes, 2-4%butanol, 20-100 mM sodium acetate, 50-70 mM citric acid, 120-130 mMsodium phosphate, 1 mM ethylene diamine tetraacetic acid (EDTA), and 1mM dithiothreitol (DTT). These agents are prepared in buffers and areadded dropwise in various combinations to the crystallization buffer.Proteins to be crystallized can be modified, e.g., by phosphorylation orby using a phosphate mimic (e.g., tungstate, cacodylate, or sulfate).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, and the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the polypeptide andprecipitant concentrations, thereby causing the polypeptide to reachsupersaturation levels.

In the hanging drop technique (McPherson, 1976), an initial polypeptidemixture is created by adding a precipitant to a concentrated polypeptidesolution. The concentrations of the polypeptide and precipitants aresuch that in this initial form, the polypeptide does not crystallize. Asmall drop of this mixture is placed on a glass slide that is invertedand suspended over a reservoir of a second solution. The system is thensealed. Typically, the second solution contains a higher concentrationof precipitant or other dehydrating agent. The difference in theprecipitant concentrations causes the protein solution to have a highervapor pressure than the second solution. Since the system containing thetwo solutions is sealed, equilibrium is established, and water from thepolypeptide mixture transfers to the second solution. This equilibriumincreases the polypeptide and precipitant concentration in thepolypeptide solution. At the critical concentration of polypeptide andprecipitant, a crystal of the polypeptide may form.

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

Yet another method of crystallization is an electrocrystallizationmethod in which use is made of the dipole moments of proteinmacromolecules that self-align in the Helmholtz layer adjacent to anelectrode (see, e.g., U.S. Pat. No. 5,597,457, the disclosure of whichis incorporated herein by reference in its entirety).

Some proteins may be recalcitrant to crystallization. However, severaltechniques are available to the skilled artisan to inducecrystallization. For example, the removal of flexible polypeptidesegments at the amino or carboxyl terminal end of the protein mayfacilitate production of crystalline protein samples. Removal of suchsegments can be done using molecular biology techniques or treatment ofthe protein with proteases such as trypsin, chymotrypsin, or subtilisin.

In diffraction experiments, a narrow and parallel beam of x-rays istaken from the x-ray source and directed onto the crystal to producediffracted beams. The incident primary beams cause damage to both themacromolecule and solvent molecules. The crystal is, therefore, cooled(e.g., to between −220° C. and −50° C.) to prolong its lifetime. Theprimary beam must strike the crystal from many directions to produce allpossible diffraction spots, so the crystal is rotated in the beam duringthe experiment. The diffracted spots are recorded on a film or by anelectronic detector. Exposed film has to be digitized and quantified ina scanning device, whereas the electronic detectors feed the signalsthey detect directly into a computer. Electronic area detectorssignificantly reduce the time required to collect and measurediffraction data. Each diffraction beam, which is recorded as a spot onfilm or a detector plate, is defined by three properties: the amplitude,which is measured from the intensity of the spot; the wavelength, whichis set by the x-ray source; and the phase, which is lost in x-rayexperiments. All three properties are needed for all of the diffractedbeams in order to determine the positions of the atoms giving rise tothe diffracted beams. One way of determining the phases is calledMultiple Isomorphous Replacement (MIR), which requires the introductionof exogenous x-ray scatterers (e.g., heavy atoms such metal atoms) intothe unit cell of the crystal. For a more detailed description of MIR,see U.S. Pat. No. 6,093,573 (column 15) the disclosure of which isincorporated herein by reference in its entirety.

Atomic coordinates refer to Cartesian coordinates (x, y, and zpositions) derived from mathematical equations involving Fouriersynthesis of data derived from patterns obtained via diffraction of amonochromatic beam of x-rays by the atoms (scattering centers) ofbiological macromolecule of interest in crystal form. Diffraction dataare used to calculate electron density maps of repeating units in thecrystal (unit cell). Electron density maps are used to establish thepositions (atomic coordinates) of individual atoms within a crystal'sunit cell. The absolute values of atomic coordinates convey spatialrelationships between atoms because the absolute values ascribed toatomic coordinates can be changed by rotational and/or translationalmovement along x, y, and/or z axes, together or separately, whilemaintaining the same relative spatial relationships among atoms. Thus, abiological macromolecule (e.g., a protein) whose set of absolute atomiccoordinate values can be rotationally or translationally adjusted tocoincide with a set of prior determined values from an analysis ofanother sample is considered to have the same atomic coordinates asthose obtained from the other sample.

Further details on x-ray crystallography can be obtained from co-pendingU.S. Application No. 2005/0015232, U.S. Pat. No. 6,093,573,PCT/US99/18441, PCT/US99/11913, and PCT/US00/03745. The disclosures ofall these patent documents are incorporated herein by reference in theirentirety.

NMR Spectroscopy.

Whereas x-ray crystallography requires single crystals of amacromolecule of interest, NMR measurements are carried out in solutionunder near physiological conditions. However, NMR-derived structures arenot as detailed as crystal-derived structures.

While the use of NMR spectroscopy was until relatively recently limitedto the elucidation of the 3-D structure of relatively small molecules(e.g., proteins of 100-150 amino acid residues), recent advancesincluding isotopic labeling of the molecule of interest and transverserelaxation-optimized spectroscopy (TROSY) have allowed the methodologyto be extended to the analysis of much larger molecules, e.g., proteinswith a molecular weight of 110 kDa (Wider, 2000).

NMR uses radio-frequency radiation to examine the environment ofmagnetic atomic nuclei in a homogeneous magnetic field pulsed with aspecific radio frequency. The pulses perturb the nuclear magnetizationof those atoms with nuclei of nonzero spin. Transient time domainsignals are detected as the system returns to equilibrium. Fouriertransformation of the transient signal into a frequency domain yields aone-dimensional NMR spectrum. Peaks in these spectra represent chemicalshifts of the various active nuclei. The chemical shift of an atom isdetermined by its local electronic environment. Two-dimensional NMRexperiments can provide information about the proximity of various atomsin the structure and in three dimensional space. Protein structures canbe determined by performing a number of two- (and sometimes 3- or 4-)dimensional NMR experiments and using the resulting information asconstraints in a series of protein folding simulations.

More information on NMR spectroscopy including detailed descriptions ofhow raw data obtained from an NMR experiment can be used to determinethe 3-D structure of a macromolecule can be found in: Protein NMRSpectroscopy, Principles and Practice, (1996); Gronenborn et al. (1990);and Wider (2000), supra., the disclosures of all of which areincorporated herein by reference in their entirety

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of compounds of the disclosure that arepeptides. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation “motif” that is substantially the same asthe three-dimensional conformation of a selected peptide. The peptidemotif provides the peptidomimetic compound with the ability to inhibitthe interaction of VLA-4 and VEGR2. Peptidomimetic compounds can haveadditional characteristics that enhance their in vivo utility, such asincreased cell permeability and prolonged biological half-life. Thepeptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

IV. THERAPIES

A. Pharmaceutical Formulations and Routes of Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present disclosure comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous media. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present disclosure, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. Of particular interest isdirect intratumoral administration, perfusion of a tumor, oradministration local or regional to a tumor, for example, in the localor regional vasculature or lymphatic system, or in a resected tumor bed.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present disclosure maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present disclosure may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences,” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, purity and general safety standards as required by FDAOffice of Biologics Standards.

B. Treatment Methods Involving Inhibition

It is envisioned that a peptide that contains binding sites for bothVEGFR2 and VLA-4, such as 210-236, has the capacity to bridge bothreceptors and link them together. This activates signaling that thecells use for migration and invasion. The inventors work (FIGS. 6A-H)demonstrates that 210-236 displays such activity with an EC₅₀ of 0.3 μM.At 30-100-fold higher concentrations, e.g., 10-30 μM, same peptide wouldcompete with itself such that it would occupy only single receptorsusing one or the other binding motif; it would not couple the receptorstogether even though it contained both binding sites, and would preventendogenous Sdc1 from the binding sites and coupling the receptors, thusacting as effective inhibitors of the mechanism. The inventors haveshown that 210-236 acts as such an inhibitor with an IC₅₀ of 10 μM.Administration of peptide such that serum levels of peptide reach eitherthe EC₅₀ or IC₅₀ concentration would be used to manipulate theactivating or inhibitory activity.

1. Cancer

Cancer cells to which the methods of the present disclosure can beapplied include generally any cell that expresses VEGFR2 and/or VLA-4,and more particularly, that overexpresses VEGFR2 and/or VLA-4. Anappropriate cancer cell can be a breast cancer, lung cancer, coloncancer, pancreatic cancer, renal cancer, stomach cancer, liver cancer,bone cancer, hematological cancer (e.g., leukemia or lymphoma), myeloma(including multiple myeloma), neural tissue cancer, melanoma, ovariancancer, testicular cancer, prostate cancer, cervical cancer, vaginalcancer, or bladder cancer cell. In addition, the methods of thedisclosure can be applied to a wide range of species, e.g., humans,non-human primates (e.g., monkeys, baboons, or chimpanzees), horses,cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils,hamsters, rats, and mice.

Peptides or analogs that inhibit Sdc1 engagement to VLA-4, or VLA-4engagement of VEGFR2 can be administered to mammalian subjects (e.g.,human breast cancer patients) alone or in conjunction with other drugsand/or radiotherapy. The compounds can also be administered to subjectsthat are genetically and/or environmentally (due to, for example,physiological and/or environmental factors) susceptible to cancer, e.g.,subjects with a family history of cancer (e.g., breast cancer), subjectswith chronic inflammation or subject to chronic stress, or subjects thatare exposed to natural or non-natural environmental carcinogenicconditions (e.g., excessive exposure to sunlight, industrialcarcinogens, or tobacco smoke).

When the methods are applied to subjects with cancer, prior toadministration of a compound, the cancer can optionally be tested forVEGFR2 and/or VLA-4 expression or overexpression by methods known in theart. In this way, subjects can be identified as being susceptible totreatments according to the present disclosure. Such methods can beperformed in vitro on cancer cells obtained from a subject.Alternatively, in vivo imaging techniques using, for example,radiolabeled antibodies specific for VEGFR2 and/or VLA-4 can beperformed.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.0001 mg/kg-100 mg/kg.Wide variations in the needed dosage are to be expected in view of thevariety of compounds available and the differing efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,5-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more times). Encapsulation ofthe polypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

2. Pathologic Wound Healing

Wound healing is an essential process in maintaining health. However, incertain instances, wound healing can create health problems. Theseinclude hypertrophic scarring, keloid or dermoid formation, andexuberant granulation. These conditions are often supported bypathologic angiogenesis (discussed below). The present disclosure may beapplied to address these conditions.

i. Keloids

A keloid is a type of scar that, depending on its maturity, is composedmainly of either type III (early) or type I (late) collagen. It is aresult of an overgrowth of granulation tissue (collagen type 3) at thesite of a healed skin injury which is then slowly replaced by collagentype 1. Keloids are firm, rubbery lesions or shiny, fibrous nodules, andcan vary from pink to flesh-coloured or red to dark brown in colour. Akeloid scar is benign, non-contagious, but sometimes accompanied bysevere itchiness and pain, and changes in texture. In severe cases, itcan affect movement of skin.

Keloids should not be confused with hypertrophic scars, which are raisedscars that do not grow beyond the boundaries of the original wound.Keloids expand in claw-like growths over normal skin. They have thecapability to hurt with a needle-like pain or to itch without warning,although the degree of sensation varies from patient to patient.

If the keloid becomes infected, it may ulcerate. Removing the scar isone treatment option; however, it may result in more severeconsequences: the probability that the resulting surgery scar will alsobecome a keloid is high, usually greater than 50%. Laser treatment hasalso been used with varying degrees of success.

Keloids form within scar tissue. Collagen, used in wound repair, tendsto overgrow in this area, sometimes producing a lump many times largerthan that of the original scar. Although they usually occur at the siteof an injury, keloids can also arise spontaneously. They can occur atthe site of a piercing and even from something as simple as a pimple orscratch. They can occur as a result of severe acne or chickenpoxscarring, infection at a wound site, repeated trauma to an area,excessive skin tension during wound closure or a foreign body in awound. Keloids can sometimes be sensitive to chlorine. Keloid scars cangrow, if they appear at a younger age, because the body is stillgrowing.

Histologically, keloids are fibrotic tumors characterized by acollection of atypical fibroblasts with excessive deposition ofextracellular matrix components, especially collagen, fibronectin,elastin, and proteoglycans. Generally, keloids contain relativelyacellular centers and thick, abundant collagen bundles that form nodulesin the deep dermal portion of the lesion. Keloids present a therapeuticchallenge that must be addressed, as these lesions can cause significantpain, pruritus (itching), and physical disfigurement. They may notimprove in appearance over time and can limit mobility if located over ajoint.

Keloids affect both sexes equally, although the incidence in youngfemale patients has been reported to be higher than in young males,probably reflecting the greater frequency of earlobe piercing amongwomen. There is a fifteen times higher frequency of occurrence in highlypigmented people. Persons of African descent are at increased risk ofkeloid occurrences.

The best treatment is prevention in patients with a knownpredisposition. This includes preventing unnecessary trauma or surgery(including ear piercing, elective mole removal), whenever possible. Anyskin problems in predisposed individuals (e.g., acne, infections) shouldbe treated as early as possible to minimize areas of inflammation.

Intra-Lesional Corticosteroids.

Intra-lesional corticosteroids are first-line therapy for most keloids.A systematic review found that up to 70 percent of patients respond tointra-lesional corticosteroid injection with flattening of keloids,although the recurrence rate is high in some studies (up to 50 percentat five years). While corticosteroids are one of the more commontreatments, injections into and in close proximity to keloid tissue canbe highly painful and can produce undesirable results in femalepatients, as per any other testosterone-based treatment.

Excision.

Scalpel excision may be indicated if injection therapy alone isunsuccessful or unlikely to result in significant improvement. Excisionshould be combined with preoperative, intraoperative, or postoperativetriamcinolone or interferon injections. Recurrence rates from 45 to 100percent have been reported in patients treated with excision alone; thisfalls to below 50 percent in patients treated with combination therapy.

Gel Sheeting.

Both hydrogel and silicone gel sheeting have been used for the treatmentof symptoms (e.g., pain and itching) in patients with establishedkeloids as well as for the management of evolving keloids and theprevention of keloids at the sites of new injuries. While the precisemechanism of action is still poorly understood, there is evidence thatapplication of gel sheeting may reduce the incidence of abnormalscarring. A controlled study found significant changes in growth factorlevels of fibronectin and IL-8 with application of hydrogel sheetingwith respect to normal skin. Silicone sheeting was associated withchanging growth factor levels of only fibronectin.

Cryosurgery.

Cryosurgery is most useful in combination with other treatments forkeloids. The major side effect is permanent hypopigmentation, whichlimits its use in people with darker skin.

Radiation Therapy.

Most studies, but not all, have found radiation therapy to be highlyeffective in reducing keloid recurrence, with improvement rates of 70 to90 percent when administered after surgical excision. A small randomizedtrial of treatments after surgery found recurrences in two of sixteenearlobe keloids (13 percent) treated with radiation therapy and in fourof twelve earlobe keloids (33 percent) treated with steroid injections.However, concern regarding the potential long-term risks (e.g.,malignancy) associated with using radiation for an essentially benigndisorder limits its utility in most patients. Only a few cases ofmalignancy that may have been associated with radiation therapy forkeloids have been reported. Although causation cannot be confirmed inthese cases, caution should still be used when prescribing radiationtherapy for keloids, particularly when treating younger patients.Radiation therapy may occasionally be appropriate as treatment forkeloids that are resistant to other therapies. In addition, radiationtherapy may be indicated for lesions that are not amenable to resection.

Interferon Alpha.

Interferon alpha injections may reduce recurrence rates postoperatively.However, all currently available studies of interferon therapy sufferfrom methodologic problems, making an evidence-based recommendationregarding its use difficult.

Pulsed Dye Laser.

Pulsed dye laser treatment can be beneficial for keloids, and appears toinduce keloid regression through suppression of keloid fibroblastproliferation, and induction of apoptosis and enzyme activity.Combination treatment with pulsed dye laser plus intralesional therapywith corticosteroids and/or fluorouracil may be superior to eitherapproach alone.

ii. Hypertrophic Scarring

Hypertrophic scars are a cutaneous condition characterized by depositsof excessive amounts of collagen which gives rise to a raised scar, butnot to the degree observed with keloids. Like keloids, they form mostoften at the sites of pimples, body piercings, cuts and burns. Theyoften contain nerves and blood vessels. They generally develop afterthermal or traumatic injury that involves the deep layers of the dermisand express high levels of TGF-β.

When a normal wound heals the body produces new collagen fibers at arate which balances the breakdown of old collagen. Hypertrophic scarsare red and thick and may be itchy or painful. They do not extend beyondthe boundary of the original wound but may continue to thicken for up to6 months. They usually improve over the one or two years but may causedistress due to their appearance or the intensity of the itching, alsorestricting movement if they are located close to a joint.

Hypertrophic scars are more common in the young and people with darkerskin. Some people have an inherited tendency to this type of scarring.It is not possible to completely prevent hypertrophic scars, so anyonewho has suffered one should inform their doctor or surgeon if they needto have surgery. Scar therapies are available which may speed up theprocess of change from a hypertrophic scar to a flatter, paler one.Scars do not occur in younger people as often as older people becausetheir skin cells replicate more quickly and fill in the wound withnormal skin tissue.

iii. Proud Flesh

Granulation tissue is the perfused, fibrous connective tissue thatreplaces a fibrin clot in healing wounds. Granulation tissue typicallygrows from the base of a wound and is able to fill wounds of almost anysize it heals. In addition, it is also found in ulcers like esophagealulcer; however, when the granulation becomes uncontrolled, oftenresulting from improper wound care, a condition known as exuberantgranulation or “proud flesh” results. The scar tissue, if untreated, maycompletely overtake the wound area. Caught early, the condition can betreated by topical or injected steroids, but more advanced cases requiresurgical intervention. Horses are subject to this disease, particularlyin the legs. Also, some individuals of African decent have a geneticpredisposition to exuberant scarring.

3. Pathologic Angiogenesis

Despite the abundancy of angiogenic factors present in differenttissues, endothelial cell turnover in a healthy adult organism isremarkably low with a turnover in the order of thousands of days. Themaintenance of endothelial quiescence is thought to be due to thepresence of endogenous negative regulators. Moreover, positive andnegative regulators often co-exist in tissues with extensiveangiogenesis. These observations have led to the hypothesis thatactivation of the endothelium depends on a balance between theseopposing regulators. If positive angiogenic factors dominate, theendothelium will be activated. Thus, the angiogenic process can bedivided in an activation phase (initiation and progression of theangiogenic process) and a phase of resolution (termination andstabilization of the vessels). It is not yet clear whether theresolution phase is due to upregulation of endogenous inhibitors orexhaustion of positive regulators.

With respect to activated endothelium, an important distinction must bemade between physiological and pathological settings. Although manypositive and negative regulators operate in both, endothelial cellproliferation is tightly controlled in the former, whereas in thelatter, the uncontrolled growth of microvessels may lead to several“angiogenic diseases” in different tissues, such as hemangiomas,psoriasis, Kaposi's sarcoma, ocular neovascularization, rheumatoidarthritis, endometriosis, atherosclerosis, tumor growth and metastasis,myocardial ischemia, peripheral ischemia, cerebral ischemia, woundhealing, reconstructive surgery, and ulcer healing, and these may alsobe advantageously treated with the compositions of the presentdisclosure. Some of these are discussed in greater detail below.

Hemangiomas are angiogenic diseases, characterized by the proliferationof capillary endothelium with accumulation of mast cells, fibroblastsand macrophages. They represent the most frequent tumors of infancy,occurring more frequently in females than males (3:1 ratio). Hemangiomasare characterized by rapid neonatal growth (proliferating phase). By theage of 6 to 10 months, the hemangioma's growth rate becomes proportionalto the growth rate of the child, followed by a very slow regression forthe next 5 to 8 years (involuting phase). Most hemangiomas occur assingle tumors whereas about 20% of the affected infants have multipletumors, which may appear at any body site. Approximately 5% producelife-, sight-, or limb-threatening complications, with high mortalityrates. The pathogenesis of hemangiomas has not yet been elucidated.However, several immunohistochemical studies have provided insight intothe histopathology of these lesions. In particular, proliferatinghemangiomas express high levels of proliferating cell nuclear antigen(PCNA, a marker for cells in the S phase), type IV collagenase, VEGF andFGF-2. During the involuting phase of hemangiomas, expression of theseangiogenic factors decreases. Furthermore, urinary levels of FGF-2 areelevated during the proliferating phase of hemangioma, but become normalduring involution or after therapy with IFN-α.

Other proliferative disorders of the skin include psoriasis and Kaposi'ssarcoma. Hypervascular psoriatic lesions express high levels of theangiogenic inducer IL-8, whereas the expression of the endogenousinhibitor TSP-1 is decreased. Kaposi's sarcoma (KS) is the most commontumor associated with human immunodeficiency virus (HIV) infection andis in this setting almost always associated with human herpes virus 8(HHV-8) infection. Typical features of KS are proliferatingspindle-shaped cells, considered to be the tumor cells and endothelialcells forming blood vessels. KS is a cytokine-mediated disease, highlyresponsive to different inflammatory mediators like IL-10, TNF-α andIFN-γ and angiogenic factors. In particular, FGF-2 was found tosynergize with HIV-tat to promote angiogenesis and KS development.Finally, growth of KS, both in vitro and in vivo, could be blocked by anantisense oligonucleotide targeting FGF-2.

Diabetic retinopathy is the leading cause of blindness in the workingpopulation, but ocular neovascularization can also occur upon exposureof preterm babies to oxygen. It is assumed that both forms are inducedby hypoxia in the retina. Elevated levels of the hypoxia-inducibleangiogenic factor VEGF were detected in the aqueous and vitreous of eyeswith proliferative retinopathy.

Excessive production of angiogenic factors from infiltratingmacrophages, immune cells or inflammatory cells may also trigger theformation of pannus, an extensively vascularized tissue that invades anddestroys the cartilage, as seen in rheumatoid arthritis. Moreover,uncontrolled angiogenesis may underlie various female reproductivedisorders, such as prolonged menstrual bleeding or infertility, andexcessive endothelial cell proliferation has been observed in theendometrium of women with endometriosis.

Angiogenesis also contributes to atherosclerosis, a major cause of deathof Western populations. Atherosclerosis is the main cause of heartattack. The walls of the coronary artery are normally free ofmicrovessels except in the atherosclerotic plaques, where there aredense networks of capillaries, known as the vasa vasorum. These fragilemicrovessels can cause hemorrhages, leading to blood clotting, with asubsequent decreased blood flow to the heart muscle and heart attack.Finally, angiogenesis is thought to be indispensable for solid tumorgrowth and metastasis.

4. Inflammatory and Autoimmune Diseases

In another aspect, the inventors contemplate the treatment of variousinflammatory and autoimmune diseases with the peptide agents describedherein. Non-limiting examples of such diseases are set forth below.

i. Inflammatory Bowel Disease

Ulcerative Colitis.

Ulcerative colitis is a disease that causes inflammation and sores,called ulcers, in the lining of the large intestine. The inflammationusually occurs in the rectum and lower part of the colon, but it mayaffect the entire colon. Ulcerative colitis rarely affects the smallintestine except for the end section, called the terminal ileum.Ulcerative colitis may also be called colitis or proctitis. Theinflammation makes the colon empty frequently, causing diarrhea. Ulcersform in places where the inflammation has killed the cells lining thecolon; the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although it canalso occur in the mouth, esophagus, stomach, duodenum, large intestine,appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop the disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies. Theories about what causes ulcerative colitis abound, but nonehave been proven. The most popular theory is that the body's immunesystem reacts to a virus or a bacterium by causing ongoing inflammationin the intestinal wall. People with ulcerative colitis haveabnormalities of the immune system, but doctors do not know whetherthese abnormalities are a cause or a result of the disease. Ulcerativecolitis is not caused by emotional distress or sensitivity to certainfoods or food products, but these factors may trigger symptoms in somepeople.

The most common symptoms of ulcerative colitis are abdominal pain andbloody diarrhea. Patients also may experience fatigue, weight loss, lossof appetite, rectal bleeding, and loss of body fluids and nutrients.About half of patients have mild symptoms. Others suffer frequent fever,bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitismay also cause problems such as arthritis, inflammation of the eye,liver disease (hepatitis, cirrhosis, and primary sclerosingcholangitis), osteoporosis, skin rashes, and anemia. No one knows forsure why problems occur outside the colon. Scientists think thesecomplications may occur when the immune system triggers inflammation inother parts of the body. Some of these problems go away when the colitisis treated.

A thorough physical exam and a series of tests may be required todiagnose ulcerative colitis. Blood tests may be done to check foranemia, which could indicate bleeding in the colon or rectum. Bloodtests may also uncover a high white blood cell count, which is a sign ofinflammation somewhere in the body. By testing a stool sample, thedoctor can detect bleeding or infection in the colon or rectum. Thedoctor may do a colonoscopy or sigmoidoscopy. For either test, thedoctor inserts an endoscope—a long, flexible, lighted tube connected toa computer and TV monitor—into the anus to see the inside of the colonand rectum. The doctor will be able to see any inflammation, bleeding,or ulcers on the colon wall. During the exam, the doctor may do abiopsy, which involves taking a sample of tissue from the lining of thecolon to view with a microscope. A barium enema x ray of the colon mayalso be required. This procedure involves filling the colon with barium,a chalky white solution. The barium shows up white on x-ray film,allowing the doctor a clear view of the colon, including any ulcers orother abnormalities that might be there.

Treatment for ulcerative colitis depends on the seriousness of thedisease. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon. Surgery is theonly cure for ulcerative colitis. Some people whose symptoms aretriggered by certain foods are able to control the symptoms by avoidingfoods that upset their intestines, like highly seasoned foods, rawfruits and vegetables, or milk sugar (lactose). Each person mayexperience ulcerative colitis differently, so treatment is adjusted foreach individual. Emotional and psychological support is important. Somepeople have remissions—periods when the symptoms go away—that last formonths or even years. However, most patients' symptoms eventuallyreturn. This changing pattern of the disease means one cannot alwaystell when a treatment has helped. Some people with ulcerative colitismay need medical care for some time, with regular doctor visits tomonitor the condition.

The goal of therapy is to induce and maintain remission, and to improvethe quality of life for people with ulcerative colitis. Several types ofdrugs are available. Aminosalicylates are drugs that contain5-aminosalicyclic acid (5-ASA), help control inflammation. Sulfasalazineis a combination of sulfapyridine and 5-ASA and is used to induce andmaintain remission. The sulfapyridine component carries theanti-inflammatory 5-ASA to the intestine. However, sulfapyridine maylead to side effects such as include nausea, vomiting, heartburn,diarrhea, and headache. Other 5-ASA agents such as olsalazine,mesalamine, and balsalazide, have a different carrier, offer fewer sideeffects, and may be used by people who cannot take sulfasalazine. 5-ASAsare given orally, through an enema, or in a suppository, depending onthe location of the inflammation in the colon. Most people with mild ormoderate ulcerative colitis are treated with this group of drugs first.Corticosteroids such as prednisone and hydrocortisone also reduceinflammation. They may be used by people who have moderate to severeulcerative colitis or who do not respond to 5-ASA drugs.Corticosteroids, also known as steroids, can be given orally,intravenously, through an enema, or in a suppository, depending on thelocation of the inflammation. These drugs can cause side effects such asweight gain, acne, facial hair, hypertension, mood swings, and anincreased risk of infection. For this reason, they are not recommendedfor long-term use. Immunomodulators such as azathioprine and6-mercapto-purine (6-MP) reduce inflammation by affecting the immunesystem. They are used for patients who have not responded to 5-ASAs orcorticosteroids or who are dependent on corticosteroids. However,immunomodulators are slow-acting and may take up to 6 months before thefull benefit is seen. Patients taking these drugs are monitored forcomplications including pancreatitis and hepatitis, a reduced whiteblood cell count, and an increased risk of infection. Cyclosporine A maybe used with 6-MP or azathioprine to treat active, severe ulcerativecolitis in people who do not respond to intravenous corticosteroids.Other drugs may be given to relax the patient or to relieve pain,diarrhea, or infection.

Occasionally, symptoms are severe enough that the person must behospitalized. For example, a person may have severe bleeding or severediarrhea that causes dehydration. In such cases the doctor will try tostop diarrhea and loss of blood, fluids, and mineral salts. The patientmay need a special diet, feeding through a vein, medications, orsometimes surgery.

About 25-40% of ulcerative colitis patients must eventually have theircolons removed because of massive bleeding, severe illness, rupture ofthe colon, or risk of cancer. Sometimes the doctor will recommendremoving the colon if medical treatment fails or if the side effects ofcorticosteroids or other drugs threaten the patient's health. Surgery toremove the colon and rectum, known as proctocolectomy, is followed byone of the following:

-   -   Ileostomy, in which the surgeon creates a small opening in the        abdomen, called a stoma, and attaches the end of the small        intestine, called the ileum, to it. Waste will travel through        the small intestine and exit the body through the stoma. The        stoma is about the size of a quarter and is usually located in        the lower right part of the abdomen near the beltline. A pouch        is worn over the opening to collect waste, and the patient        empties the pouch as needed.    -   Ileoanal anastomosis, or pull-through operation, which allows        the patient to have normal bowel movements because it preserves        part of the anus. In this operation, the surgeon removes the        diseased part of the colon and the inside of the rectum, leaving        the outer muscles of the rectum. The surgeon then attaches the        ileum to the inside of the rectum and the anus, creating a        pouch. Waste is stored in the pouch and passed through the anus        in the usual manner. Bowel movements may be more frequent and        watery than before the procedure. Inflammation of the pouch        (pouchitis) is a possible complication.        Not every operation is appropriate for every person. Which        surgery to have depends on the severity of the disease and the        patient's needs, expectations, and lifestyle. People faced with        this decision should get as much information as possible by        talking to their doctors, to nurses who work with colon surgery        patients (enterostomal therapists), and to other colon surgery        patients. Patient advocacy organizations can direct people to        support groups and other information resources.

Most people with ulcerative colitis will never need to have surgery. Ifsurgery does become necessary, however, some people find comfort inknowing that after the surgery, the colitis is cured and most people goon to live normal, active lives.

Crohn's Disease.

Another disorder for which immunosuppression has been tried is Crohn'sdisease. Crohn's disease symptoms include intestinal inflammation andthe development of intestinal stenosis and fistulas; neuropathy oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective. Immunosuppression withcyclosporine is sometimes beneficial for patients resistant to orintolerant of corticosteroids. Nevertheless, surgical correction iseventually required in 90% of patients; 50% undergo colonic resection.The recurrence rate after surgery is high, with 50% requiring furthersurgery within 5 years.

One hypothesis for the etiology of Crohn's disease is that a failure ofthe intestinal mucosal barrier, possibly resulting from geneticsusceptibilities and environmental factors (e.g., smoking), exposes theimmune system to antigens from the intestinal lumen including bacterialand food antigens. Another hypothesis is that persistent intestinalinfection by pathogens such as Mycobacterium paratuberculosis, Listeriamonocytogenes, abnormal Escherichia coli, or paramyxovirus, stimulatesthe immune response; or alternatively, symptoms result from adysregulated immune response to ubiquitous antigens, such as normalintestinal microflora and the metabolites and toxins they produce. Thepresence of IgA and IgG anti-Sacccharomyces cerevisiae antibodies (ASCA)in the serum was found to be highly diagnostic of pediatric Crohn'sdisease.

In Crohn's disease, a dysregulated immune response is skewed towardcell-mediated immunopathology. But immunosuppressive drugs, such ascyclosporine, tacrolimus, and mesalamine have been used to treatcorticosteroid-resistant cases of Crohn's disease with mixed success.

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies.In particular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease. Another approach tothe treatment of Crohn's disease has focused on at least partiallyeradicating the bacterial community that may be triggering theinflammatory response and replacing it with a non-pathogenic community.For example, U.S. Pat. No. 5,599,795 discloses a method for theprevention and treatment of Crohn's disease in human patients. Theirmethod was directed to sterilizing the intestinal tract with at leastone antibiotic and at least one anti-fungal agent to kill off theexisting flora and replacing them with different, select,well-characterized bacteria taken from normal humans. Borody taught amethod of treating Crohn's disease by at least partial removal of theexisting intestinal microflora by lavage and replacement with a newbacterial community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacteroides and Escherichiacoli species. However, there has been no known cause of Crohn's diseaseto which diagnosis and/or treatment could be directed.

Rheumatoid Arthritis.

The exact etiology of RA remains unknown, but the first signs of jointdisease appear in the synovial lining layer, with proliferation ofsynovial fibroblasts and their attachment to the articular surface atthe joint margin. Subsequently, macrophages, T cells and otherinflammatory cells are recruited into the joint, where they produce anumber of mediators, including the cytokines interleukin-1 (IL-1), whichcontributes to the chronic sequalae leading to bone and cartilagedestruction, and tumour necrosis factor (TNF-α), which plays a role ininflammation. The concentration of IL-1 in plasma is significantlyhigher in patients with RA than in healthy individuals and, notably,plasma IL-1 levels correlate with RA disease activity. Moreover,synovial fluid levels of IL-1 are correlated with various radiographicand histologic features of RA.

In normal joints, the effects of these and other proinflammatorycytokines are balanced by a variety of anti-inflammatory cytokines andregulatory factors. The significance of this cytokine balance isillustrated in juvenile RA patients, who have cyclical increases infever throughout the day. After each peak in fever, a factor that blocksthe effects of IL-1 is found in serum and urine. This factor has beenisolated, cloned and identified as IL-1 receptor antagonist (IL-1ra), amember of the IL-1 gene family. IL-1ra, as its name indicates, is anatural receptor antagonist that competes with IL-1 for binding to typeI IL-1 receptors and, as a result, blocks the effects of IL-1. A 10- to100-fold excess of IL-1ra may be needed to block IL-1 effectively;however, synovial cells isolated from patients with RA do not appear toproduce enough IL-1ra to counteract the effects of IL-1.

ii. Systemic Lupus Erythematosus

There has also been no known cause for autoimmune diseases such assystemic lupus erythematosus. Systemic lupus erythematosus (SLE) is anautoimmune rheumatic disease characterized by deposition in tissues ofautoantibodies and immune complexes leading to tissue injury (Kotzin,1996). In contrast to autoimmune diseases such as MS and type 1 diabetesmellitus, SLE potentially involves multiple organ systems directly, andits clinical manifestations are diverse and variable. For example, somepatients may demonstrate primarily skin rash and joint pain, showspontaneous remissions, and require little medication. At the other endof the spectrum are patients who demonstrate severe and progressivekidney involvement that requires therapy with high doses of steroids andcytotoxic drugs such as cyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (GN). Glomerulonephritis is a serious condition inwhich the capillary walls of the kidney's blood purifying glomerulibecome thickened by accretions on the epithelial side of glomerularbasement membranes. The disease is often chronic and progressive and maylead to eventual renal failure.

The mechanisms by which autoantibodies are induced in these autoimmunediseases remains unclear. As there has been no known cause of SLE, towhich diagnosis and/or treatment could be directed, treatment has beendirected to suppressing immune responses, for example with macrolideantibiotics, rather than to an underlying cause. (e.g., U.S. Pat. No.4,843,092).

iii. Multiple Sclerosis

Multiple sclerosis (MS), also known as disseminated sclerosis orencephalomyelitis disseminata, is an inflammatory disease in which theinsulating covers of nerve cells in the brain and spinal cord aredamaged. This damage disrupts the ability of parts of the nervous systemto communicate, resulting in a wide range of signs and symptoms,including physical, mental, and sometimes psychiatric problems. MS takesseveral forms, with new symptoms either occurring in isolated attacks(relapsing forms) or building up over time (progressive forms). Betweenattacks, symptoms may disappear completely; however, permanentneurological problems often occur, especially as the disease advances.

Multiple sclerosis is the most common autoimmune disorder affecting thecentral nervous system. As of 2008, between 2 and 2.5 million people areaffected globally with rates varying widely in different regions of theworld and among different populations. The disease usually beginsbetween the ages of 20 and 50 and is twice as common in women as in men.The name multiple sclerosis refers to scars (sclerae-better known asplaques or lesions) in particular in the white matter of the brain andspinal cord.

A person with MS can have almost any neurological symptom or sign; withautonomic, visual, motor, and sensory problems being the most common.The specific symptoms are determined by the locations of the lesionswithin the nervous system, and may include loss of sensitivity orchanges in sensation such as tingling, pins and needles or numbness,muscle weakness, very pronounced reflexes, muscle spasms, or difficultyin moving; difficulties with coordination and balance (ataxia); problemswith speech or swallowing, visual problems (nystagmus, optic neuritis ordouble vision), feeling tired, acute or chronic pain, and bladder andbowel difficulties, among others. Difficulties thinking and emotionalproblems such as depression or unstable mood are also common. Uhthoffsphenomenon, a worsening of symptoms due to exposure to higher than usualtemperatures, and Lhermitte's sign, an electrical sensation that runsdown the back when bending the neck, are particularly characteristic ofMS. The main measure of disability and severity is the expandeddisability status scale (EDSS), with other measures such as the multiplesclerosis functional composite being increasingly used in research.

The condition begins in 85% of cases as a clinically isolated syndromeover a number of days with 45% having motor or sensory problems, 20%having optic neuritis, and 10% having symptoms related to brainstemdysfunction, while the remaining 25% have more than one of the previousdifficulties. The course of symptoms occurs in two main patternsinitially: either as episodes of sudden worsening that last a few daysto months (called relapses, exacerbations, bouts, attacks, or flare-ups)followed by improvement (85% of cases) or as a gradual worsening overtime without periods of recovery (10-15% of cases). A combination ofthese two patterns may also occur or people may start in a relapsing andremitting course that then becomes progressive later on. Relapses areusually not predictable, occurring without warning. Exacerbations rarelyoccur more frequently than twice per year. Some relapses, however, arepreceded by common triggers and they occur more frequently during springand summer. Similarly, viral infections such as the common cold,influenza, or gastroenteritis increase their risk. Stress may alsotrigger an attack. Women with MS who become pregnant experience fewerrelapses; however, during the first months after delivery the riskincreases. Overall, pregnancy does not seem to influence long-termdisability. Many events have not been found to affect relapse ratesincluding vaccination, breast feeding, physical trauma, and Uhthoffsphenomenon.

The cause of MS is unknown; however, it is believed to occur as a resultof some combination of environmental factors such as infectious agentsand genetics. Theories try to combine the data into likely explanations,but none has proved definitive. While there are a number ofenvironmental risk factors and although some are partly modifiable,further research is needed to determine whether their elimination canprevent MS.

MS is more common in people who live farther from the equator, althoughexceptions exist. These exceptions include ethnic groups that are at lowrisk far from the equator such as the Samis, Amerindians, CanadianHutterites, New Zealand Maori, and Canada's Inuit, as well as groupsthat have a relatively high risk close to the equator such asSardinians, inland Sicilians, Palestinians and Parsis. The cause of thisgeographical pattern is not clear. While the north-south gradient ofincidence is decreasing, as of 2010 it is still present.

MS is more common in regions with northern European populations and thegeographic variation may simply reflect the global distribution of thesehigh-risk populations. Decreased sunlight exposure resulting indecreased vitamin D production has also been put forward as anexplanation. A relationship between season of birth and MS lends supportto this idea, with fewer people born in the northern hemisphere inNovember as compared to May being affected later in life. Environmentalfactors may play a role during childhood, with several studies findingthat people who move to a different region of the world before the ageof 15 acquire the new region's risk to MS. If migration takes placeafter age 15, however, the person retains the risk of his home country.There is some evidence that the effect of moving may still apply topeople older than 15.

MS is not considered a hereditary disease; however, a number of geneticvariations have been shown to increase the risk. The probability ishigher in relatives of an affected person, with a greater risk amongthose more closely related. In identical twins both are affected about30% of the time, while around 5% for non-identical twins and 2.5% ofsiblings are affected with a lower percentage of half-siblings. If bothparents are affected the risk in their children is 10 times that of thegeneral population. MS is also more common in some ethnic groups thanothers.

Specific genes that have been linked with MS include differences in thehuman leukocyte antigen (HLA) system—a group of genes on chromosome 6that serves as the major histocompatibility complex (MHC). That changesin the HLA region are related to susceptibility has been known for overthirty years, and additionally this same region has been implicated inthe development of other autoimmune diseases such as diabetes type I andsystemic lupus erythematosus. The most consistent finding is theassociation between multiple sclerosis and alleles of the MHC defined asDR15 and DQ6. Other loci have shown a protective effect, such asHLA-C554 and HLA-DRB 1*11. Overall, it has been estimated that HLAchanges account for between 20 and 60% of the genetic predisposition.Modern genetic methods (genome-wide association studies) have discoveredat least twelve other genes outside the HLA locus that modestly increasethe probability of MS.

Many microbes have been proposed as triggers of MS, but none have beenconfirmed. Moving at an early age from one location in the world toanother alters a person's subsequent risk of MS. An explanation for thiscould be that some kind of infection, produced by a widespread microberather than a rare one, is related to the disease. Proposed mechanismsinclude the hygiene hypothesis and the prevalence hypothesis. Thehygiene hypothesis proposes that exposure to certain infectious agentsearly in life is protective, the disease being a response to a lateencounter with such agents. The prevalence hypothesis proposes that thedisease is due to an infectious agent more common in regions where MS iscommon and where in most individuals it causes an ongoing infectionwithout symptoms. Only in a few cases and after many years does it causedemyelination. The hygiene hypothesis has received more support than theprevalence hypothesis.

Evidence for a virus as a cause include: the presence of oligoclonalbands in the brain and cerebrospinal fluid of most people with MS, theassociation of several viruses with human demyelinationencephalomyelitis, and the occurrence of demyelination in animals causedby some viral infection. Human herpes viruses are a candidate group ofviruses. Individuals having never been infected by the Epstein-Barrvirus are at a reduced risk of getting MS, whereas those infected asyoung adults are at a greater risk than those having had it at a youngerage. Although some consider that this goes against the hygienehypothesis, since the non-infected have probably experienced a morehygienic upbringing, others believe that there is no contradiction,since it is a first encounter with the causative virus relatively latein life that is the trigger for the disease. Other diseases that may berelated include measles, mumps and rubella.

Smoking has been shown to be an independent risk factor for MS. Stressmay be a risk factor although the evidence to support this is weak.Association with occupational exposures and toxins—mainly solvents—hasbeen evaluated, but no clear conclusions have been reached. Vaccinationswere studied as causal factors; however, most studies show noassociation. Several other possible risk factors, such as diet andhormone intake, have been looked at; however, evidence on their relationwith the disease is “sparse and unpersuasive”. Gout occurs less thanwould be expected and lower levels of uric acid have been found inpeople with MS. This has led to the theory that uric acid is protective,although its exact importance remains unknown.

Multiple sclerosis is typically diagnosed based on the presenting signsand symptoms, in combination with supporting medical imaging andlaboratory testing. It can be difficult to confirm, especially early on,since the signs and symptoms may be similar to those of other medicalproblems. The McDonald criteria, which focus on clinical, laboratory,and radiologic evidence of lesions at different times and in differentareas, is the most commonly used method of diagnosis with the Schumacherand Poser criteria being of mostly historical significance. While theabove criteria allow for a non-invasive diagnosis, some state that theonly definitive proof is an autopsy or biopsy where lesions typical ofMS are detected.

Clinical data alone may be sufficient for a diagnosis of MS if anindividual has had separate episodes of neurologic symptomscharacteristic of the disease. In those who seek medical attention afteronly one attack, other testing is needed for the diagnosis. The mostcommonly used diagnostic tools are neuroimaging, analysis ofcerebrospinal fluid and evoked potentials. Magnetic resonance imaging ofthe brain and spine may show areas of demyelination (lesions orplaques). Gadolinium can be administered intravenously as a contrastagent to highlight active plaques and, by elimination, demonstrate theexistence of historical lesions not associated with symptoms at themoment of the evaluation. Testing of cerebrospinal fluid obtained from alumbar puncture can provide evidence of chronic inflammation in thecentral nervous system. The cerebrospinal fluid is tested foroligoclonal bands of IgG on electrophoresis, which are inflammationmarkers found in 75-85% of people with MS. The nervous system in MS mayrespond less actively to stimulation of the optic nerve and sensorynerves due to demyelination of such pathways. These brain responses canbe examined using visual- and sensory-evoked potentials.

Secondary progressive MS occurs in around 65% of those with initialrelapsing-remitting MS, who eventually have progressive neurologicdecline between acute attacks without any definite periods of remission.Occasional relapses and minor remissions may appear. The most commonlength of time between disease onset and conversion fromrelapsing-remitting to secondary progressive MS is 19 years.

The primary progressive subtype occurs in approximately 10-20% ofindividuals, with no remission after the initial symptoms. It ischaracterized by progression of disability from onset, with no, or onlyoccasional and minor, remissions and improvements. The usual age ofonset for the primary progressive subtype is later than of therelapsing-remitting subtype. It is similar to the age that secondaryprogressive usually begins in relapsing-remitting MS, around 40 years ofage. Progressive relapsing MS describes those individuals who, fromonset, have a steady neurologic decline but also have clear superimposedattacks. This is the least common of all subtypes.

Unusual types of MS have been described; these include Devic's disease,Balo concentric sclerosis, Schilder's diffuse sclerosis, and Marburgmultiple sclerosis. There is debate on whether they are MS variants ordifferent diseases. Multiple sclerosis behaves differently in children,taking more time to reach the progressive stage. Nevertheless, theystill reach it at a lower average age than adults usually do.

Although there is no known cure for multiple sclerosis, severaltherapies have proven helpful. The primary aims of therapy are returningfunction after an attack, preventing new attacks, and preventingdisability. As with any medical treatment, medications used in themanagement of MS have several adverse effects. Alternative treatmentsare pursued by some people, despite the shortage of supporting evidence.

During symptomatic attacks, administration of high doses of intravenouscorticosteroids, such as methylprednisolone, is the usual therapy, withoral corticosteroids seeming to have a similar efficacy and safetyprofile. Although, in general, effective in the short term for relievingsymptoms, corticosteroid treatments do not appear to have a significantimpact on long-term recovery. The consequences of severe attacks that donot respond to corticosteroids might be treatable by plasmapheresis.

As of 2014, nine disease-modifying treatments have been approved byregulatory agencies for relapsing-remitting multiple sclerosis (RRMS)including: interferon β-1a, interferon β-1b, glatiramer acetate,mitoxantrone, natalizumab, fingolimod, teriflunomide, dimethyl fumarateand alemtuzumab. Their cost effectiveness as of 2012 is unclear.

In RRMS they are modestly effective at decreasing the number of attacks.The interferons and glatiramer acetate are first-line treatments and areroughly equivalent, reducing relapses by approximately 30%.Early-initiated long-term therapy is safe and improves outcomes.Natalizumab reduces the relapse rate more than first-line agents;however, due to issues of adverse effects is a second-line agentreserved for those who do not respond to other treatments or with severedisease. Mitoxantrone, whose use is limited by severe adverse effects,is a third-line option for those who do not respond to othermedications. Treatment of clinically isolated syndrome (CIS) withinterferons decreases the chance of progressing to clinical MS. Efficacyof interferons and glatiramer acetate in children has been estimated tobe roughly equivalent to that of adults. The role of some of the neweragents such as fingolimod, teriflunomide, and dimethyl fumarate, as of2011, is not yet entirely clear.

No treatment has been shown to change the course of primary progressiveMS and as of 2011 only one medication, mitoxantrone, has been approvedfor secondary progressive MS. In this population tentative evidencesupports mitoxantrone moderately slowing the progression of the diseaseand decreasing rates of relapses over two years.

D. Treatments Involving Agonism

In another embodiment, the disclosure contemplates enhancing theengagement of VLA-4 and VEGFR2 with syndecan-1 or active fragmentsthereof. Peptides having this activity would need, at a minimum, tocontain the binding site for both of these molecules, which are foundbetween residues 210 and 236 of syndecan-1. Any shorter molecules, suchas 210-233 (SEQ ID NO: 4) or 214-236 (SEQ ID NO: 7), would be incapableof agonism. In particular, this agonism would be applied to endothelialcells that express both VLA-4 and VEGFR2.

Wound healing, or wound repair, is an intricate process in which theskin (or another organ-tissue) repairs itself after injury. In normalskin, the epidermis (outermost layer) and dermis (inner or deeper layer)exists in a steady-state equilibrium, forming a protective barrieragainst the external environment. Once the protective barrier is broken,the normal (physiologic) process of wound healing is immediately set inmotion. The classic model of wound healing is divided into three or foursequential yet overlapping phases: (1) hemostasis, (2) inflammatory, (3)proliferative and (4) remodeling. Upon injury to the skin, a set ofcomplex biochemical events takes place in a closely orchestrated cascadeto repair the damage. Within minutes post-injury, platelets(thrombocytes) aggregate at the injury site to form a fibrin clot. Thisclot acts to control active bleeding (hemostasis).

In the inflammatory phase, bacteria and debris are phagocytosed andremoved, and factors are released that cause the migration and divisionof cells involved in the proliferative phase.

The proliferative phase is characterized by angiogenesis, collagendeposition, granulation tissue formation, epithelialization, and woundcontraction. In angiogenesis, new blood vessels are formed by vascularendothelial cells. In fibroplasia and granulation tissue formation,fibroblasts grow and form a new, provisional extracellular matrix (ECM)by excreting collagen and fibronectin. Concurrently,re-epithelialization of the epidermis occurs, in which epithelial cellsproliferate and ‘crawl’ atop the wound bed, providing cover for the newtissue.

In contraction, the wound is made smaller by the action ofmyofibroblasts, which establish a grip on the wound edges and contractthemselves using a mechanism similar to that in smooth muscle cells.When the cells' roles are close to complete, unneeded cells undergoapoptosis.

In the maturation and remodeling phase, collagen is remodeled andrealigned along tension lines and cells that are no longer needed areremoved by apoptosis. However, this process is not only complex butfragile, and susceptible to interruption or failure leading to theformation of chronic non-healing wounds. Factors which may contribute tothis include diabetes, venous or arterial disease, old age, andinfection. The phases of wound healing normally progress in apredictable, timely manner; if they do not, healing may progressinappropriately to either a chronic wound such as a venous ulcer orpathological scarring such as a keloid scar.

Treatment of wounds depends on how severe the wound is, its location,and whether other areas are affected. If another condition is causingproblems with wound healing, it is important to treat or control thisproblem. A caregiver may prescribe antibiotics to fight infection,either orally, i.v., or applied directly on the wound area. Palliativecare such as for pain, swelling and fever are often prescribed. Woundcare is essential as well and includes cleansing, debridement and wounddressing. Dressings are particularly important to protect the wound fromfurther injury and infection. These may also help give pressure todecrease swelling. Dressings may be in the form of bandages, films, orfoams. They may contain certain substances that may help promote fasterhealing. Sometimes, skin taken from another part of the body may be usedto close a large wound. The skin may also be man-made, which containsspecial cells needed to repair damaged tissues. Additional treatmentsinclude hyperbaric oxygen therapy (HBO), negative pressure therapy (alsocalled vacuum-assisted closure or “VAC”), or creams, ointments, ormedicines with special solutions which help in wound healing may beapplied to the wound.

V. COMBINATION THERAPIES

A. Cancer

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. In the contextof the present disclosure, it is contemplated that syndecan peptidetherapy could be used similarly in conjunction with chemotherapeutic,radiotherapeutic, or immunotherapeutic intervention.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentdisclosure, one would generally contact a target cell with a syndecanpeptide and at least one other therapy. These therapies would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theagents/therapies at the same time. This may be achieved by contactingthe cell with a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell with two distinctcompositions or formulations, at the same time, wherein one compositionincludes the syndecan peptide and the other includes the agent.

Alternatively, the syndecan treatment may precede or follow the othertreatment by intervals ranging from minutes to weeks. In embodimentswhere the other treatment and the syndecan peptide are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the therapies would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would contact the cell with both modalities within about 12-24 hoursof each other, within about 6-12 hours of each other, or with a delaytime of only about 12 hours. In some situations, it may be desirable toextend the time period for treatment significantly; however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) pass between the respective administrations.

It also is conceivable that more than one administration of either thesyndecan peptide or the other therapy will be desired. Variouscombinations may be employed, where the syndecan peptide is “A,” and theother therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. Again, to achieve cell killing,both therapies are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy include anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic” or “genotoxic agents,”are intended to be of use in the combined treatment methods disclosedherein. In treating cancer according to the disclosure, one wouldcontact the tumor cells with an agent in addition to the expressionconstruct. This may be achieved by irradiating the localized tumor sitewith radiation such as X-rays, UV-light, γ-rays or even microwaves.Alternatively, the tumor cells may be contacted with the agent byadministering to the subject a therapeutically effective amount of apharmaceutical composition.

Various classes of chemotherapeutic agents are comtemplated for use within combination with peptides of the present disclosure. For example,selective estrogen receptor antagonists (“SERMs”) include Tamoxifen,4-hydroxy Tamoxifen (Afimoxfene), Falsodex, Raloxifene, Bazedoxifene,Clomifene, Femarelle, Lasofoxifene, Ormeloxifene, and Toremifene.

Chemotherapeutic agents contemplated to be of use, include, e.g.,camptothecin, actinomycin-D, mitomycin C. The disclosure alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. The agent may beprepared and used as a combined therapeutic composition, or kit, bycombining it with peptides, as described above.

Agents that directly cross-link DNA or form adducts are also envisaged.Agents such as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include Adriamycin, also known as Doxorubicin, Etoposide,Verapamil, Podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for Doxorubicin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally. Microtubuleinhibitors, such as taxanes, also are contemplated. These molecules arediterpenes produced by the plants of the genus Taxus, and includepaclitaxel and docetaxel.

mTOR, the mammalian target of rapamycin, also known as FK506-bindingprotein 12-rapamycin associated protein 1 (FRAP1) is a serine/threonineprotein kinase that regulates cell growth, cell proliferation, cellmotility, cell survival, protein synthesis, and transcription. Rapamycinand analogs thereof (“rapalogs”) are therefore contemplated for use incombination cancer therapy in accordance with the present disclosure.

Another possible combination therapy with the peptides claimed herein isTNF-α (tumor necrosis factor-alpha), a cytokine involved in systemicinflammation and a member of a group of cytokines that stimulate theacute phase reaction. The primary role of TNF is in the regulation ofimmune cells. TNF is also able to induce apoptotic cell death, to induceinflammation, and to inhibit tumorigenesis and viral replication.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, x-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors affect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for x-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventor proposes that the local or regional delivery of syndecanpeptides to patients with cancer will be a very efficient method fortreating the clinical disease. Similarly, the chemo- or radiotherapy maybe directed to a particular, affected region of the subject's body.Alternatively, regional or systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

In addition to combining syndecan therapies with chemo- andradiotherapies, combinations with immunotherapy, hormone therapy, toxintherapy and surgery are also contemplated. In particular, one may employtargeted therapies such as Avastin, Erbitux, Gleevec, Herceptin andRituxan.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating cancer.

B. Prevention of Scarring/Abberrant Wound Healing

In other embodiments, one may use peptides of the present disclosure incombination with other therapies to prevent scarring. These includecorticosteroids, laser therapy, cryosurgery and interferon ca.

C. Inflammatory and Autoimmune Diseases

Inflammatory and autoimmune diseases may be treated in combinationsincluding peptides of the present disclosure and other agents, such assteroids, NSAIDs, anti-inflammatory cytokines, or any other agentsmentioned as first line therapies for these conditions.

D. Promoting of Wound Healing

In the context of the present disclosure, it is contemplated thatsyndecan-1 or a fragment retaining the ability to facilitate VLA-4interaction with VEGFR2 may be used in combination with a secondtherapeutic agent to more effectively treat wounds. Additionaltherapeutic agents contemplated for use in combination with syndencan-1or active fragments thereof include, but are not limited to other woundhealing agents, protective agents, and scar reducing agents and thelike. Specific examples include corticosteroids, cytotoxic drugs,antibiotics, antiseptics, nicotine, anti-platelet drugs, NSAIDS,colchicines, anti-coagulants, vasoconstricting drugs andimmunosuppressives, as well as HBO and VAC methods, discussed above.

To aid in the wound healing process, using the methods and compositionsof the present disclosure, one would generally contact a cell withsyndecan-1 or an active fragment thereof in combination with a secondagent. These compositions would be provided in a combined amounteffective to exert a combined effect on the damaged tissue. This processmay involve contacting the cells with syndecan-1 or active fragment, incombination with a second therapeutic agent or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the syndencan-1 oractive fragment and the other includes the second agent.

Alternatively, treatment with syndecan-1 or active fragments, or saltsor analogs thereof, may precede or follow the additional agent treatmentby intervals ranging from minutes to weeks. In embodiments where thesecond agent is applied separately to the target, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the agent would still be able to exert anadvantageously combined effect on the target. In such instances, it iscontemplated that one would contact the target with both modalitieswithin about 12-24 hr of each other and, more preferably, within about6-12 hr of each other. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It also is conceivable that more than one administration of the peptidesdescribed herein in combination with a second therapeutic agent will bedesired. Various combinations may be employed, where the syndencan-1 orfragment is “A” and the second therapeutic agent is “B”, as exemplifiedbelow:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated.

In the present disclosure, a number of drugs or agents may proveparticularly useful when combined with syndencan-1. Such agents/drugsinclude corticosteroids, NSAIDs or any other anti-inflammatory, acytotoxic drug, an antibiotic, antimicrobial, antifungal or antiseptic,nicotine, an anti-platelet drug, colchicine, anti-coagulants,vasoconstricting drugs or immunosuppressives.

VI. EXAMPLES

The following examples are included to demonstrate particularembodiments of the disclosure. It should be appreciated by those ofskill in the art that the techniques disclosed in the examples whichfollow represent techniques discovered by the inventor to function wellin the practice of the disclosure, and thus can be considered toconstitute particular modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the disclosure.

Example 1—Materials and Methods

Reagents.

SST0001, an N-acetylated glycol split heparin is a potent inhibitor ofHPSE and was kindly provided by Sigma-tau Industrie FarmaceuticheRiunite S.p.A. (Pomezia, Italy). S1ED²¹⁰⁻²⁴⁰ peptide from GeneScriptCorporation (location) was reconstituted in DME medium (Invitrogen) 200mM HEPES (pH7.4) (Sigma). VEGFR2 inhibitor Vandetanib (ZD6474) wasobtained from LC Laboratorues (Woburn, Mass., USA). VEGF165 was obtainedfrom PeproTech (Rocky Hill, N.J., USA). Recombinant GST andGST-mouse-S1ED proteins were prepared as previously described (Beauvaiset al., 2004a; 2004b). Rabbit anti-pY¹¹⁷⁵ VEGFR2 serum (mAb 19A10) andrabbit anti-VEGFR2 (mAb 55B11) were obtained from Cell SignalingTechnology (Danvers, Mass., USA). Mouse anti-human VEGFR2 serum (CH-11)and mouse anti-α4-integrin (P1H4) were obtained from Millipore.Polyclonal antibodies against human Sdc1 were affinity-purified aspreviously described (Beauvais et al., 2004a). All secondary antibodieswere purchased from Jackson ImmunoResearch (West Grove, Pa., USA). Mouseanti-human MMP-9 (clone 6-6B) antibody was from Calbiochem.

Cell Culture.

All cell lines were cultured at 37° C. with 7.5% CO₂, 92.5% air and 85%humidity. CAG myeloma cells, established at the Myeloma Institute forResearch and Therapy (Little Rock, Ark., USA), were transfected withempty vector (HPSE^(low) cells) or vector containing the cDNA for humanHPSE (HPSE^(high) cells) as described. P3×63Ag8 mouse myeloma cells werefrom the ATCC. Cells were grown in RPMI1640 (Invitrogen) with 10% fetalbovine serum (Atlanta Biologicals, Lawrenceville, Ga., USA), 4 mML-glutamine (Fisher Scientific), and 0.05 mM β-mercaptoethanol (Sigma).HMEC-1 were kindly provided by Drs. E. W. Ades and F. J. Candal (Centerfor Disease Control, Atlanta, Ga., USA) and Dr. T. J. Lawley (EmoryUniversity, Atlanta, Ga., USA. HMEC-1 cells were grown in MCDB131 medium(Mediatech, Manassas, Va., USA) supplemented with 5 mM L-glutamine, 20mM NaHCO₃, 10 ng/ml epidermal growth factor, 1 pg mL hydrocortisone,bovine brain extract with heparin and antibiotics (SingleQuot kit;Lonza, Walkersville, Md., USA) and 15% fetal bovine serum (AtlantaBiologicals, Lawrenceville, Ga., USA).

Cell Spreading Assay.

Nitrocellulose-coated slides were coated for 2 hr at 37° C. with 40μg/ml FN (kindly provided by Dr. Donna Peters, University ofWisconsin-Madison) or 5 μg/ml recombinant VCAM-1/Fc chimera (R&DSystems) in calcium and magnesium-free PBS (CMF-PBS; 135 mM NaCl, 2.7 mMKCl, 10.2 mM Na₂HPO₄-7H₂O and 1.75 mM KH₂PO₄, pH7.4). Slides wereblocked for 1 hr at 37° C. with RPMI 1640 containing 1% heat-denaturedBSA (plating medium). Cells in plating medium were allowed to attach andspread for 2.5 hr at 37° C. Slides were fixed in 4% EM gradeparaformaldehyde (Electron Microscopy Sciences) in CMF-PBS and labeledfor 30 min in 0.13 μM rhodamine phalloidin (Invitrogen) in CMF-PBS.Coverslips were mounted in non-fluorescing mounting medium (Immumount;Thermo Shandon) and allowed to dry. All images were acquired with aNikon Microphot FX microscope using a 20× objective (Nikon; Ex 541-551,DM 580, Barrier 590), Photometric CoolSnap ES camera, and version7.7.3.0 Metamorph© Imaging software (Molecular Devices). All imagesrepresent results from triplicate wells and at least three independentexperiments.

Immunostaining.

Fixed cells on FN-coated slides were quenched with 0.1 M glycine,permeabilized with CMF-PBS containing 0.1% Triton X-100 for 3 min atroom temperature (RT) and blocked with 5% BSA in CMF-PBS. Cells werestained with primary antibodies to α4-integrin (P1H4), VEGFR2 (mAb55B11), VEGFR2 (CH-11) or GST for 2 hr at RT, rinsed in CMF-PBS,incubated for 1 hr with secondary antibodies (Alexa-488-conjugated goatanti-mouse IgG (H+L) F(ab′)₂ and Alexa-546-conjugated goat anti-rabbitIgG (H+L) F(ab′)₂ secondary antibody (Molecular Probes) (1:100 dilution)in blocking buffer, followed by washing in CMF-PBS and double-distilledH₂O before mounting in Immumount. All images represent results fromtriplicate wells and at least three independent experiments. Images wereprocessed and colorized using Adobe Photoshop (Adobe Systems).

Migration Assay.

The bottom chambers of Transwell filter chambers (8 μm pores; Corning)were coated with 40 μg/ml of FN. Cells (5×10⁵ in 0.2 ml plating medium)were placed in the upper chamber and incubated for 16 hr at 37° C. inthe CO₂ incubator. After incubation, cells in the upper chamber wereremoved and cells on the bottom side of the filter were fixed in 4%paraformaldehyde (PFA) and stained with 0.1% Crystal Violet beforeimaging. Cells from at least five random fields were counted.

Time-Lapse Live Cell Imaging.

HPSE^(low) cells or HPSE^(high) cells were plated in FN-coated 48-wellplates in plating medium and allowed to attach and spread for 2.5 hr.After washing, attached cells were observed at 37° C. on a Nikon EclipseTE2000U microscopy system equipped with environment chamber using aPlanApo 20× objective (0.75 numerical aperture) and a PhotometricsCoolSnap ES camera. Cells were tracked with Metamorph and Images werecollected at 10 min intervals over 4 hr.

Immunoprecipitation.

HPSE^(low) cells were plated on FN in the presence of 4 μg/ml GST orGST-S1ED for 2.5 hr. The cells were washed with CMF-PBS, then lysed for20 min on ice in lysis buffer (0.5% Triton X-100 in 50 mM Hepes, 50 mMNaCl and 10 mM EDTA (pH 7.4) containing a 1:1000 dilution of proteaseinhibitor mixture set III (Calbiochem). Cell lysates were precleared at13000 rpm for 20 min at 4° C. using 50 μg/ml isotype-matched nonspecificIgG and 30 μl of protein G agarose (GE Healthcare), then incubated at 4°C. overnight with either 10 μg/ml anti-α4-integrin (P1H4) or VEGFR2 (mAb55B11) or nonspecific mouse IgG. Immunoprecipitated α4-integrin orVEGFR2 were dissolved in SDS sample buffer), electrophoresed andanalyzed on western blots as described previously. Visualization ofimmunoreactive bands was performed using ECF reagent (AmershamPharmacia) to detect alkaline-phosphatae-conjugated probing antibody andscanned on a Typhoon Trio Variable Mode Imager (GE Healthcare).

GST Pull Down Assay.

CAG cells were washed with CMF-PBS and then lysed for 20 min on ice inlysis buffer. Cell debris was removed by centrifugation at 13000 rpm for20 min at 4° C. Whole cell lysates were then incubated at 4° C.overnight with either 3 μM GST or 3 μM GST-S1ED in the absence orpresence of 1 μM, 3 μM, and 10 μM S1ED²¹⁰⁻²⁴⁰ peptide. GST or GST-S1EDwas captured by addition of glutathione Sepharose and the beads werewashed with ice-cold lysis buffer and CMF-PBS, prior to addition ofSDS-PAGE sample buffer and analyzed by electrophoresis and westernblotting as described previously.

Example 2—Results

Adhesion and Spreading of CAG Cells on FN or VCAM-1 is Enhanced by HPSE.

Myeloma cells expressing high levels of HPSE are more invasive thancells expressing low levels of the enzyme (Yang et al., 2002; Mahtouk etal., 2007 and Yang et al., 2005). To test whether such cells alsodisplay increased adhesion to FN and VCAM-1, cell adhesion ligandsexpressed in the myeloma tumor niche, CAG myeloma cells transfected withthe cDNA for HPSE and expressing high levels of the enzyme (HPSE^(high))were compared to cells transfected with empty vector alone that expresslow endogenous levels (HPSE^(low)). When plated onto FN or VCAM-1, bothcell types were observed to attach equally (FIGS. 1A-B). However,HPSE^(high) cells spread rapidly and form a polarized morphology,whereas the majority of the HPSE^(low) cells do not (FIGS. 1A-B). Toconfirm that the spreading of HPSE^(high) cells is attributed to HPSEexpression, HPSE was inhibited by pre-treatment with the HPSE inhibitorSST0001 (Ritchie et al., 2011). This treatment blocked HPSE^(high) cellspreading on FN and VCAM-1, but had no effect on attachment of eithercell type (FIGS. 1A-B).

FN and VCAM-1 are ligands for the α4-integrin (VLA-4), which isexpressed on myeloma cells (Sanz-Rodriguez et al., 1999). Thus, theinventors questioned whether VLA-4 mediates the adhesion and spreadingthat they observe. Blocking of VLA-4 with a function-blocking antibody,clone P1H4, resulted in complete inhibition of cell attachment of bothHPSE^(high) and HPSE^(low) cells (FIG. 1B).

Many cell types, including myeloma cells, that constitutively expresshigh levels of HPSE respond over time with altered gene expression, dueboth to enzymatic and nonenzymatic activities of HPSE (Zetser et al.,2003; Levy-Adam et al., 2008; Goldshmidt et al., 2003 and Sotnikov etal., 2004). To test whether the polarized phenotype of the HPSE^(high)cells was due to altered gene expression in response to long-termconstitutive expression of this enzyme, or was a more immediate responseto trimming of heparan sulfate (HS) chains during the adhesion andspreading event, HPSE^(low) and HPSE^(high) cells were treated for 2 hrwith heparinase III (HPIII), a bacterial enzyme that completely degradesthe HS (Desai et al., 1993), before plating on FN and VCAM-1.Destruction of the HS by HPIII induces spreading of HPSE^(low) cellscomparable to that observed for HPSE^(high) cells (FIG. 1C). In sum,these data suggest that trimming of HS chains during VLA-4-mediatedadhesion to FN and VCAM-1 activates a cell polarization response in theCAG cells. The bipolar morphology displayed by the HPSE^(high) cells istypical of motile cells (Ridley, 2011; Ridley et al., 2003). To testthis, the inventors examined cell migration towards FN in serum-freemedium using a Transwell migration assay (Keely, 2001). HPSE^(high)cells efficiently migrated toward FN, whereas HPSE^(low) cells failed todo so (FIG. 1D).

Shed Sdc1 Mediates the HPSE-Enhanced Effect in CAG Cells.

Trimming of the HS chains on Sdc1 by HPSE induces MMP9-mediated shedding(Yang et al., 2007 and Ramani et al., 2012). To test whether Sdc1shedding might have a role in the HPSE-induced motility of the CAGcells, HPSE^(low) and HPSE^(high) cells were plated on VCAM-1 in theabsence or presence of an MMP9 blocking antibody. The blocking antibodyeffectively blocks the polarized spreading of HPSE^(high) cells onVCAM-1 without disrupting adhesion (FIG. 2A). To confirm that Sdc1 isshed in response to MMP9 cleavage, HPSE^(low) and HPSE^(high) cells weregrown in suspension at equal densities with or without 10 μg/ml MMP9blocking antibody (clone 6-6B). After 48 hr, the conditioned media wereharvested and shed Sdc1 was visualized on a western blot (FIG. 2B).Higher amounts of Sdc1 were detected in the conditioned medium ofHPSE^(high) cells compared to HPSE^(low) cells, but is reduced tocontrol levels by MMP9 blocking antibody (FIG. 2B).

To test whether the shed Sdc1 protein or HS chains are responsible forthe altered phenotype of the HPSE^(high) CAG cells, the inventorsquestioned whether recombinant GST-tagged Sdc1 ectodomain (GST-S1ED)expressed in bacteria and devoid of HS could mimic the effect of MMP9cleavage. GST-S1ED was added to HPSE^(high) cells in the presence orabsence of MMP9 blocking antibody. As expected, the spreading of theHPSE^(high) cells is blocked in the presence of MMP9 blocking antibody.However, the polarized phenotype is rescued by GST-S1ED addition (FIG.2C), even in the presence of the MMP9 inhibitor.

These findings suggest that the Sdc1 ectodomain contains an active sitethat promotes the invasive phenotype. To identify this putative site,deletions and truncations were introduced into the GST-S1ED constructand the mutant proteins were tested in the spreading assay usingHPSE^(low) cells plated on VCAM-1 (FIG. 3A). Full length GST-SLED,GST-S1ED¹⁷⁵⁻²⁴⁹, GST-S1ED¹⁷⁵⁻²⁴⁰, GST-S1ED²¹⁰⁻²⁴⁹ and GST-S1ED²⁰⁰⁻²⁴⁰induced cell spreading. However, GST-S1ED¹⁷⁻¹⁹⁵, GST-S1ED¹⁷⁵⁻¹⁹⁴ andGST-S1ED¹⁷⁵⁻²¹² did not, implicating a putative active site spanningamino acids 210-240 (FIGS. 3A-B). Testing this prediction by deletion ofamino acids 210-240 (GST-S1ED^(Δ210-240)) also abolishes the ability ofGST-S1ED to induce cell spreading, confirming this as the active region(FIG. 3B). To test if this region is not only necessary, but alsosufficient to induce the invasive phenotype, HPSE^(low) cells weretreated with a peptide corresponding to amino acids 210-240,S1ED²¹⁰⁻²⁴⁰. As shown in FIG. 3C, the S1ED²¹⁰⁻²⁴⁰ peptide is sufficientto induce HPSE^(low) cell spreading on VCAM-1. Further truncation ofthis peptide demonstrates that S1ED²¹⁰⁻²³⁶ retains full activity,whereas S1ED²¹⁰⁻²³³ or S1ED²¹⁴⁻²⁴⁰ do not (FIG. 3C). Thus, the aminoacids 210-213 and 234-236 are critical for the invasive phenotype(highlighted in red in FIG. 3B).

HPSE Expression Leads to Activation of VEGFR2 when VLA-4 Engages Ligand.

Next, the inventors questioned how the shed Sdc1 promotes the myelomacell invasive phenotype. They have shown previously that Sdc1 acts as anorganizer for other cell surface receptors, including receptor tyrosinekinases (RTKs) (Rapraeger et al., 2013; Wang et al. 2010; Beauvais andRapraeger, 2010). The inventors hypothesized that amino acids 210-236 inthe ectodomain of Sdc1 may be a site that organizes and activates RTKs.To test this hypothesis, they treated HPSE^(high) cells with various RTKinhibitors to identify an RTK responsible for this phenotype. They foundthat blockade of vascular endothelial growth factor receptor-2 (VEGFR2)kinase activity with Vandetanib completely inhibits HPSE^(high) cellspreading on FN (FIG. 4A). This treatment did not affect the adhesion ofeither the HPSE^(low) or HPSE^(high) cells (FIG. 4A). CAG cells areknown to produce VEGF as an autocrine growth factor and it has beenshown to promote the disease (Kumar et al., 2003; Giatromanolaki et al.,2010; Purushothaman et al., 2010). To test whether activation of VEGFR2by exogenous VEGF also promotes polarized spreading of the cells, VEGFwas added to HPSE^(low) cells plated on FN. Surprisingly, addition ofVEGF failed to induce spreading of the cells. Likewise, blocking VEGFbinding to VEGFR2 with a blocking antibody failed to block the invasivephenotype observed in the HPSE^(high) cells (FIG. 4A). In sum, VEGFR2activation appears to be critical for the altered phenotype but VEGFR2activation is independent of VEGF, suggesting a novel Sdc1-dependentmechanism for activation of VEGFR2 in the myeloma cells.

The inventors next questioned whether VEGFR2 is upstream (e.g.,responsible for shedding) or downstream (e.g., a target of the shedSdc1) of Sdc1 shedding by testing whether or not Sdc1 mimetic peptidecould rescue Vandetinib-blocked spreading. HPSE^(high) cell spreading onFN was inhibited using either anti-MMP9 blocking antibody or Vandetanibin the presence or absence of S1ED²¹⁰⁻²³⁶. As observed earlier (cf.FIGS. 3A-C), S1ED²¹⁰⁻²³⁶ rescued spreading in the presence of MMP9inhibitor (FIG. 4B). However, it fails to rescue spreading blocked byVandetanib (FIG. 4B), suggesting that VEGFR2 activity is required for astep downstream of MMP9-induced Sdc1 shedding. To directly test whetheror not VEGFR2 is a target that is activated by Sdc1, HPSE^(low) cellswere suspended or plated on FN in the presence or absence ofS1ED²¹⁰⁻²³⁶. VEGFR2 activation was assessed by measuringautophosphorylation at Tyr1054/1059 in its kinase domain (Lamalice etal., 2007). When HPSE^(low) cells are treated with the peptide while insuspension, there is no alteration of VEGFR2 phosphorylation (FIG. 4C,left). However, S1ED²¹⁰⁻²³⁶ peptide causes VEGFR2 phosphorylation inHPSE^(low) cells plated on FN (FIG. 4C, left), to which they adhere viaVLA-4 (cf, FIG. 1B).)

S1ED 210-236 Causes Capture of VEGFR2 by VLA-4.

Having established that S1ED²¹⁰⁻²³⁶ induces VEGFR2 activation in anadhesion-dependent manner, the question remains why VEGFR2 activationdepends on shed Sdc1 and VLA-4-mediated adhesion. One possibility isthat shed Sdc1 couples VEGFR2 to VLA-4 that is clustered to sites of FNor VCAM-1 engagement, thereby clustering and activating VEGFR2 bytransphosphorylation. To test this, HPSE^(high) cells were plated on FN,then immunostained for VLA-4 and VEGFR2. The inventors found that theintegrin and VEGFR2 co-localize on the protrusive lamellipodium (FIG.5A). Next, they tested whether this co-localization would be induced bySdc1 extracellular domain. For this, HPSE^(low) cells were plated on FNin the presence of GST-S1ED to induced polarized spreading, then wereimmunostained for VLA-4, VEGFR2 and the GST tag on S1ED to see where theS1ED was localized. As shown in FIG. 5B, GST-S1ED co-localized withVEGFR2 or VLA-4 at the protrusive lamellipodium. The Sdc1-mediatedassociation of VEGFR2 with VLA-4 was further confirmed byimmunoprecipitation (FIG. 5C). VEGFR2 immunoprecipitated from HPSE^(low)cells plated on FN failed to precipitate VLA-4. However, VLA-4 isco-immunoprecipitated with VEGFR2 from HPSE^(low) cells plated on FN inthe presence of GST-S1ED.

The Inhibitory Peptides Block the HPSE-Induced Invasive Phenotype.

As a final confirmation of the role of Sdc1 in the HPSE-induced invasivephenotype, the inventors tested whether or not high concentrations ofthe S1ED²¹⁰⁻²⁴⁰ peptide would block the HPSE-induced invasive phenotypeof HPSE^(high) cells. As proof of principle, the effect of S1ED²¹⁰⁻²⁴⁰peptide was further tested on cell migration through a Transwell filtercoated with FN. FN-induced migration in HPSE^(high) cells was decreasedby treatment with 30 μM S1ED²¹⁰⁻²⁴⁰. Although the peptide inducedmigration of HPSE^(low) cells at low concentrations (e.g., 0.3-3 μM), itfailed to induce at 30 μM, suggesting that it inactivates rather thanactivates the mechanism at this concentration. To define which aminoacids within this peptide are necessary to activate and inhibit the HPSEinduced invasive phenotype, three peptides containing truncations fromthe N- or C-terminus were tested, e.g., S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³,S1ED²¹⁴⁻²⁴⁰ (shown in FIG. 6B). Only S1ED²¹⁰⁻²³⁶ induces HPSE^(low) cellspreading equal to that observed with S1ED²¹⁰⁻²⁴⁰, suggesting that itretains the minimal sequence for this activity (FIG. 6D) and confirmingthe results shown in FIG. 3C These data suggest that amino acids 234-236(PVD) and 210-213 (DFTF) (shown in red in FIG. 6B) are necessary toactivate the mechanism. Interestingly, S1ED²¹⁰⁻²³⁶ and S1ED²¹⁰⁻²³³ blockcell adhesion at high concentrations (30 μM), suggesting that theydisrupt VLA-4 affinity or avidity. This inhibition is independent ofVEGFR2 and does not depend on Sdc1 shedding, since the Sdc1 is not shedin the absence of HPSE expression, nor is VEGFR2 active on these cells.This suggests that these peptides potentially block VLA-4 activity evenon cells that are not being induced to invade by HPSE. S1ED²¹⁴⁻²⁴⁰,which lacks the N-terminal DFTF sequence, does not cause cell spreadingor block adhesion, suggesting that the DFTF sequence that it lacks maybe specific for binding VLA-4.

Turning to the HPSE^(high) cells, which are already induced to spread byshed Sdc1, the inventors found that S1ED²¹⁰⁻²³⁶ and S1ED²¹⁰⁻²³³ blockcell spreading when added at increasing concentrations, but theseconcentrations also induce cell detachment, suggesting that they affectVLA-4-mediated adhesion and making it difficult to assess whether theyspecifically disrupt the spreading mechanism (FIG. 6E). However, it isreadily observed that S1ED²¹⁴⁻²⁴⁰ blocks the spreading of HPSE^(high)cells because it has no effect on cell adhesion (FIG. 6E). Regardless ofmechanism, these three peptides disrupt the adhesion and/or invasivephenotype of the HPSE^(high) cells. This is confirmed by testinginvasion directly on FN-coated transwell filters (FIG. 6F). WhereasHPSE^(high) cells are observed to migrate through the filter,S1ED²¹⁰⁻²³⁶, S1ED²¹⁰⁻²³³ and S1ED²¹⁴⁻²⁴⁰ all reduce migration by 60-80%,similar to inhibition of VEGFR2 signaling using vandetanib (FIG. 6F).

To test whether the different abilities of the peptides to inhibitadhesion and/or spreading reflected their abilities to interact withVLA-4 or VEGFR2, they were used as competitors during capture of VEGFR2or VLA-4 by GST-S1ED (FIG. 6G). GST-S1ED captures both VLA-4 and VEGFR2when incubated with CAG myeloma cell lysates. S1ED²¹⁰⁻²³⁶ effectivelyblocks capture of both receptors when used at 30 μM. In contrast,S1ED²¹⁴⁻²⁴⁰ only competes for capture of VEGFR2, confirming that theN-terminal DFTF sequence is necessary for binding VLA-4 and competingwith its capture by SLED. Conversely, S1ED²¹⁰⁻²³³ competes for captureof VEGFR2, identifying the C-terminal PVD motif as the VEGFR2 capturesite.

Similar studies were conducted with P3×63Ag8 mouse myeloma cells (P3×cells) in order to further test the inhibitory properties of S1ED²¹⁰⁻²³³(SEQ ID No: 4) and S1ED²¹⁴⁻²³⁶ (SEQ ID No: 7), peptides that lack eitherthe VLA-4 or VEGFR2 binding sites, respectively. As expected,S1ED²¹⁰⁻²³³ blocks the adhesion of the myeloma cells to FN, whereasS1ED²¹⁴⁻²³⁶ is without effect (FIG. 7A). However, the invasion of theP3X cells through filters coated with FN is blocked by either peptide(FIG. 7B), confirming the requirement for both VLA-4 activation andVEGFR2-coupling to active VLA4 by Sdc1 to acquire the invasivephenotype. Treatment with inhibitors confirms that this invasion dependson HPSE, VLA-4 and VEGFR2 activation (FIG. 7B).

VLA-4 and VEGFR2 are normally expressed on vascular endothelial andlymphatic cells, suggesting that the mechanism observed in myeloma ispart of a normal mechanism in the vasculature. Using immortal HMEC-1vascular endothelial cells as a model system, the inventors found thatthese cells express levels of HPSE equivalent to the HPSE^(high) myelomacells (FIG. 8A). Conditioned medium from the HMEC-1 cells contains shedSdc1 and this shedding is prevented by the HPSE inhibitor SST0001 (FIG.8B). Furthermore, recombinant GST-S1ED captures VLA-4 and VEGFR2 fromHMEC-1 cells lysates (FIG. 8C), similar to capture observed in myelomacells. HMEC-1 cells plated on the IIICs fragment of FN, a specific VLA-4ligand, spread rapidly during a 2 hr spreading assay (FIG. 8D). VLA-4and Sdc1 co-localize to sites in the spreading margins of the cells, asdo Sdc1 and VEGFR2, or VLA-4 and VEGFR2, indicating that these threereceptors form a ternary complex at these sites. Binding and spreadingis completely dependent on VLA-4, as inhibition of this integrin withVLA-4 or β1-integrin specific antibodies prevents adhesion (FIG. 8E).Furthermore, the VLA-4 dependent spreading is dependent on theHPSE-induced shedding of Sdc1 and activation of VEGFR2, as it isprevented by HPSE inhibitor, MMP-9 blocking antibody, and vandetanib, aswell as by S1ED²¹⁰⁻²³³ and S1ED²¹⁴⁻²⁴⁰ (FIG. 8E). As with the myelomacells, blocking of VEGF binding to VEGFR2 has no effect. HMEC-1 cellsplated on IIICS activate VEGFR2, as observed by monitoring pY1175 onwestern blots, and this activation is disrupted by 10 M S1ED peptidesexpected to block the invasive phenotype. This is confirmed by transwellinvasion assays on Fn coated filters, in which blockade of VLA-4 (P1H4antibody), inhibition of HPSE (SST0001), blockade of Sdc1 shedding(anti-MMP9), blockade of VEGFR2 activation (vandetanib) or S1ED²¹⁰⁻²³³,S1ED²¹⁴⁻²⁴⁰ or S1ED²¹⁰⁻²³⁶ all block invasion (FIG. 8G). Last, theinventors tested the ability of the peptides to block angiogenesis usingthe in vitro tube formation assay (FIG. 8H). HMEC-1 cells plated onmatrigel containing 100 μg/ml FN overnight form the typical honeycombnetwork of endothelial tubules. Tube formation on this matrix is notenhanced by VEGF. Inhibition of HPSE, or addition of S1ED²¹⁰⁻²³⁶,S1ED²¹⁰⁻²³³, or S1ED²¹⁴⁻²⁴⁰, blocks tube formation, demonstrating a roleof the Sdc1-coupled VEGFR2 mechanism in angiogenesis.

As a final test of peptide stability and efficacy in vivo, theS1ED²¹⁰⁻²⁴⁰ peptide (called SSTN₂₁₀₋₂₄₀ because it is being used as aninhibitor), which inhibits the mechanism at 30 μM concentrations, wasdelivered systemically to mice bearing human breast cancer (SKBr3)xenografts at a concentration estimated to reach 30 μM in the blood. Thepeptide effectively reduced the size of the tumors (FIG. 9), potentiallyby blocking the VLA-4/VEGFR2 mechanism necessary for tumor-inducedangiogenesis upon which these tumor rely.

Example 3—Discussion

Multiple myeloma is an incurable cancer in which high expression of shedSdc1 is linked to poor outcome. Expression of HPSE, which trims the HSchains on Sdc1 leading to increasing expression of shed Sdc1 leads toaggressive disease and poor outcome. However, the molecular mechanismsunderlying the bioactivity of shed Sdc1 remain largely unknown. In thepresent study, the inventors reveal that shed Sdc1 activates VEGFR2 topromote an invasive phenotype. Mechanistically, the native, membraneanchored Sdc1 engages VLA-4 and is required for it to form high affinityadhesions with its ligands, the matrix ligand FN or stromalcell/endothelial cell receptor VCAM-1. When Sdc1 is shed, it acquiresthe additional ability to mediate VEGFR2 interaction with VLA-4clustered to these adhesion sites, leading to VEGFR2 activation. Thisrequires VLA-4 clustering to these adhesion sites. This traces to anactive site, aa210-236, in the Sdc1 ectodomain that can be mimicked by ashort peptide encompassing this sequence, or can be inhibited forshorter peptides comprising parts of this sequence that either interactwith VLA-4 alone and block adhesion, or interact with VEGFR2 alone andblock its activation.

HPSE acts as a tumor promoter via enzymatic as well as non-enzymaticmeans. However, its enzymatic activity is required for the invasivephenotype described here. Accumulating evidence suggests that suchtrimming may de-protect Sdc1 for recognition by other proteins (Ramaniet al., 2012). In the current example, the trimming appears to exposeSdc1 to MMP9, shown previously to cause shedding of the proteoglycanfrom the surface of HPSE-expressing myeloma cells when its HS chains aretrimmed (Purushothaman et al., 2008; Ramani et al., 2012). ThisHPSE-stimulated release of bioactive Sdc1 has been shown to promotetumor growth and metastasis in vivo (Yang et al., 2002 and Yang et al.,2007). In the mechanism defined here, the inventors find that the shedSdc1 activates VEGFR2 in a ligand-independent manner; although VEGFR2 istypically expressed on endothelial cells, its expression is aberrantlyupregulated in a number of tumor types, including myeloma (Kumar et al.,2003).

Another key feature of this mechanism is VLA-4. VLA-4 participates inmyeloma cell adhesion to ILDV (Ile-leu-Asp-Val) and QIDS(Gln-Ile-Asp-Ser) motifs in FN and VCAM-1, respectively (Noborio-Hatanoet al., 2009; Sanz-Rodriguiez et al., 1999; Michigami et al., 2000; Imaiet al., 2010; Vacca et al., 1995). Multiple myeloma is characterized bythe formation of multiple lytic lesions throughout the skeleton,suggesting dissemination of myeloma cells via continuous entry into andextravasation from the microvasculature. Myeloma cells encounter VCAM-1on endothelial cells within the microvasculature during theirdissemination, and VCAM-1 and FN are found on stromal cells and in thematrix, respectively, within the bone marrow (Sanz-Rodriguez et al.,1999; Vande Broek et al., 2008). VLA-4 engagement has multiple roles inadvancement of MM, as it promotes bone resorption by osteoclasts byup-regulating the release of the osteoclast-activating cytokinesmacrophage inflammatory protein (MIP)-1α and MIP-1β (Abe et al., 2009;Michigami et al., 2000), promotes cell adhesion-mediated drug resistance(CAM-DR), which can be overcome in part by Bortezomib-induceddownregulation of integrin expression (Nororio-Hatano et al., 2009).

The findings here suggest that shed Sdc1 promotes the engagement ofVEGFR2 with matrix-bound VLA-4, leading to VEGFR2 activation. The meansby which VEGFR2 is activated may trace to integrin clustering. Theaffinity of integrins for their ligands is mediated partly by theiractivation, driven by inside-out signaling, and partly by avidity, aconsequence of active integrin clustering via interactions within theirtransmembrane domains. The inventors' finding that VEGFR2 is activatedby Sdc1 only on adherent cells, and not on cells in suspension, suggeststhat clustering of the integrin serves to cluster and activate VEGFR2 aswell. Indeed, activation of VEGFR2 by VEGF in the absence of shed Sdc1fails to cause the invasive phenotype, ostensibly because VEGFR2 is notassociated with the integrin. This suggests that the integrin, VEGFR2and the syndecan must physically be associated for activation to occur.In this regard, the activation mechanism is similar to another receptorcomplex organized by Sdc1, namely, the Sdc1-IGF-1R-αvβ3 or αvβ5 integrincomplex (Beauvais et al., 2009; Beauvais et al., 2010). IGF-1R isactivated by clustering of the complex via Sd1 to sites of matrixadhesion, leading to an IGF-1R-generated inside-out signal thatactivates the integrin. This activation does not require IGF1, althoughIGF1 can enhance the signal. However, displacement of IGF-1R from theternary receptor complex blocks its ability to activate the integrin,even if IGF-1R itself is activated by IGF1. In the current exampleinvolving VEGFR2, it appears that VLA-4 is already active due to itsengagement with Sdc1, but its coupling to VEGFR2 when Sdc1 is shed leadsto VEGFR2 activation and altered downstream signaling from theVLA-4/VEGFR2 complex that causes the highly invasive phenotype. Activeintegrin appears localized to the leading edge of a protrusivelamellipodium that characterizes these invasive cells. VEGFR2 is likelyto co-localize with the integrin at these sites when the two receptorsare coupled by shed Sdc1, a mechanism that can be recapitulated byadding exogenous recombinant GST-S1ED to HPSE^(low) myeloma cells.

Interestingly, the VEGFR2 is not activated by native, membrane-anchoredSdc1. This also sets this mechanism apart from Sdc1-coupled IGF-1R,which requires the Sdc1 to be membrane bound. Although the reason forthis is not known, the inventors speculate that release of the Sdc1 fromits membrane anchorage may be necessary for the protein to re-orientsuch that is can retain its interaction with the integrin, butsimultaneously fit a binding pocket on VEGFR2. IGF-1R coupling to humansyndecans requires a.a. 93-120 (92-119 in mouse), which is distal to themembrane proximal a.a. 210-236 site necessary for VEGFR2 activation. Itis interesting to note that bioactive juxtamembrane sites exist in othersyndecans as well. A site in Sda4 (87-131) originally described byMcFall and Rapraeger has been shown to have several highly conservedamino acids that mediate b1-integrin-dependent attachment of fibroblasts(Whiteford et al., 2008). Whiteford has also described a site in Sdc2that appears responsible for PTP activity. Whether more than one siteexists in these other syndecans, making them multifunctional organizersof receptor signaling as the inventors now describe in Sdc1, remains tobe seen.

The fact that shed Sdc1 permeates the tumor microenvironment in myelomasuggests that it may have a role in VEGFR2 activation on other celltypes. These findings suggest that it plays a prominent role duringangiogenesis mediated by vascular endothelial cells, which also expressVLA-4 integrin along with VEGFR2. Note that HPSE expression has alsobeen documented in activated endothelial cells, suggesting a potentialrole for Sdc1 shedding in VLA-4 and VEGFR2 signaling even in the absenceof tumor cells. The inventors' prior work has demonstrated a role forshed Sdc1 derived from the conditioned medium of HPSE^(high) myelomacells in the induction of angiogenesis, both in an aortic ring outgrowthassay and in HUVEC sprouting to form microvessels in matrigel. Thisactivity was dependent on VEGF, the HS on the Sdc1, and the Sdc1 coreprotein itself. At least part of the bioactivity of the core proteinappeared to trace to its role in activating IGF-1R, which is necessaryfor VEGF-stimulated VEGFR2 activity and is blocked by SSTN₉₂₋₁₁₉. Thecurrent findings suggest that the 210-236 site identified here also hasa role.

In summary, the inventors have identified a mechanism by which HPSEexpression in myeloma cells leads to activation of VEGFR2 and aninvasive phenotype. The phenotype depends on coupling VEGFR2 to VLA-4via an active site (residues 210-236) in shed Sdc1. Peptides containingthis sequence, or containing a partial sequence capable of engagingVLA-4 or VEGFR2 alone, act as potent inhibitors of this mechanism. Theycan inhibit by preventing the association of VEGFR2 with VLA-4, or, asshown for S1ED²¹⁰⁻²³³ and S1ED²¹⁰⁻²³⁶, can prevent VLA-4 fromfunctioning by preventing its engagement with Sdc1, regardless ofwhether the syndecan is shed or not. This mechanism in which VEGFR2 iscoupled to VLA-4 by Sdc1 is likely to have a role in the invasion ofmyeloma cells during intravasion and extravasation in which the cellsencounter FN in the bone marrow microenvironment, and VCAM-1 on thesurface of endothelial cells lining the vasculature or on stromal cellswith the marrow. Although described on myeloma cells, the mechanism mayalso be operative on other cancers where HPSE is known to act as a tumorpromoter, and during angiogenesis. The peptides that block the mechanismhave significant potential as therapeutics to target cancer and otherdiseases that depends on this mechanism. Furthermore, the peptides thattarget VLA-4 alone and disrupt its adhesion ability may target yet otherimmune, vascular or cancer cells where VLA-4 functions independently ofVEGFR2 and/or Sdc1.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the disclosure. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

VII. REFERENCES

The following references, to the extent they provide exemplaryprocedural or details supplementary to those set forth herein, areincorporated herein by reference:

-   Abe et al., J Bone Miner Metab 27: 16-23, 2009.-   Alexander et al., Nat. Genet., 25:329-332, 2000.-   Alon and Feigelson, Semin. Immunol. 14:93-104, 2002.-   Alon et al., J. Cell. Biol. 128:1243-53, 1995.-   Anttonen et al., Br. J. Cancer, 79:558-564, 1999.-   Baciu and Goetinck, Mol. Biol. Cell, 6:1503-1513, 1995.-   Barash et al., FEBS J, 277(19): p. 3890-903, 2010.-   Barbareschi et al., Cancer, 98:474-483, 2003.-   Bayer-Garner et al., J. Cutan. Pathol., 28:135-139, 2001.-   Beauvais and Rapraeger, J. Cell Sci., 123(Pt 21): 3796-807 (2010).-   Beauvais and Rapraeger, Reprod Biol Endocrinol, 2: p. 3, 2004.-   Beauvais et al., J Cell Biol, 167(1): p. 171-81, 2004a.-   Beauvais et al., J Exp Med, 206(3): p. 691-705, 2009.-   Beauvais et al., J. Cell Biol., Reprod. Biol. Endocrinol, 2:3,    2004b.-   Bemfield et al., Annu. Rev. Biochem., 68:729-777, 1999.-   Bemfield et al., Annu. Rev. Cell Biol., 8:365-393, 1992.-   Bodanszky et al., J. Antibiot., 29(5):549-53, 1976.-   Burbach et al., Matrix Biol., 22:163-177, 2003.-   Carey et al., Exp. Cell Res., 214:12-21, 1994a.-   Carey et al., J. Cell Biol., 124:161-170, 1994b.-   Cohen et al., J. Med. Chem., 33:883-894, 1990.-   Conejo et al., Int. J. Cancer, 88:12-20, 2000.-   Couchman et al., Int. Rev. Cytol., 207:113-150, 2001.-   Crescimanno et al., J. Pathol., 189:600-608, 1999.-   Damiano and Dalton, Leuk Lymphoma 38: 71-81, 2000.-   Damiano et al., Blood 93: 1658-1667, 1999.-   David et al., J. Cell Biol., 118(4):961-969, 1992.-   Desai et al., Biochemistry, 32(32): p. 8140-5, 1993.-   Fujiya et al., Jpn. J. Cancer Res., 92:1074-1081, 2001.-   Garmy-Susini et al., Proc. Nat'l Acad. Sci. USA 110:9042-9047, 2013.-   Garmy-Susini et al., Cancer Res. 70:3042-51, 2010.-   Garmy-Susini et al., J. Clin Invest. 115:1542-51, 2005.-   Giatromanolaki et al., Anticancer Res, 30(7): p. 2831-6, 2010.-   Goldshmidt et al., FASEB J, 17(9): p. 1015-25, 2003.-   Granes et al., Exp. Cell Res., 248:439-456, 1999.-   Gronenborn et al., Anal. Chem., 62(1):2-15, 1990.-   Hansen et al., J. Cell Biol., 126:811-819, 1994.-   Hirabayashi et al., Tumour Biol., 19:454-463, 1998.-   Iba et al., J. Cell Biol., 149:1143-1156, 2000.-   Imai et al., Int J Hematol 91: 569-575, 2010.-   Inki and Jalkanen, Ann. Med., 28:63-67, 1996.-   Izzard et al., Exp. Cell Res., 165:320-336, 1986.-   Jackson, Seminars in Oncology, 24:L164-172, 1997.-   Johnson et al., In: Biotechnology And Pharmacy, Pezzuto et al.    (Eds.), Chapman and Hall, NY, 1993.-   Jones et al., J. Med. Chem., 39:904-917, 1996.-   Kato et al., Mol. Biol. Cell, 6:559-576, 1995.-   Keely, Methods Enzymol, 333: p. 256-66, 2001.-   Kelly et al., Cancer Res, 63(24): p. 8749-56, 2003.-   Khan et al., J. Biol. Chem., 263:11314-113148, 1988.-   Khotskaya et al., J Biol Chem, 284(38): p. 26085-95, 2009.-   Kim et al., Mol. Biol. Cell, 5:797-805, 1994.-   Klass et al., J. Cell Sci., 113:493-506, 2000.-   Klatka, Eur. Arch. Otorhinolaryngol., 259:115-118, 2002.-   Kumar et al., Leukemia, 17(10): p. 2025-31, 2003.-   Kumar-Singh et al., J. Pathol., 186:300-305, 1998.-   Lamalice et al., Circ Res, 100(6): p. 782-94, 2007.-   Laubach et al., Med Oncol, 27 Suppl 1: p. S1-6, 2010.-   Lebakken and Rapraeger, J. Cell Biol., 132:1209-1221, 1996.-   Leppa et al. Cell Regul., 2:1-11, 1991.-   Leppa et al., J. Cell Sci., 109:1393-1403, 1996.-   Leppa et al., Proc. Natl. Acad. Sci. USA, 89:932-936, 1992.-   Levy et al., Br. J. Cancer, 74:423-431, 1996.-   Levy et al., Bull. Cancer, 84:235-237, 1997.-   Levy-Adam et al., PLoS One, 3(6): p. e2319, 2008.-   Levy-Adam et al., Semin Cancer Biol, 20(3): p. 153-60, 2010.-   Liu et al., J. Biol. Chem., 273:22825-22832, 1998.-   Mahtouk et al., Blood, 109(11): p. 4914-23, 2007.-   Martinelli et al., Haematologica, 86(9): p. 908-17, 2001.-   Matsumoto et al., Int. J. Cancer, 74:482-491, 1997.-   McFall and Rapraeger, J. Biol. Chem., 272:12901-12904, 1997.-   McFall and Rapraeger, J. Biol. Chem., 273:28270-28276, 1998.-   McPherson, J. Biol. Chem., 251:6300-6306, 1976.-   McQuade et al., J Cell Sci, 119(Pt 12): p. 2445-56, 2006.-   Meads et al., Clin Cancer Res, 14(9): p. 2519-26, 2008.-   Merrifield, J. Am. Chem. Soc., 85:2149-2154, 1963.-   Michigami et al., Blood 96: 1953-1960, 2000.-   Miranti and Brugge, Nat. Cell Biol., 4:E83-90, 2002.-   Mundhenke et al., Am. J. Pathol., 160:185-194, 2002.-   Nakaerts et al., Int. J Cancer, 74:335-345, 1997.-   Nakanishi et al., Intl. J. Cancer, 80:527-532, 1999.-   Navia et al., Curr. Opin. Struct. Biol., 2:202-210, 1992.-   Noborio-Hatano et al., Oncogene 28: 231-242, 2009.-   Numa et al., Int. J. Oncol., 20:39-43, 2002.-   O'Connell et al., Am J Clin Pathol, 121(2): p. 254-63, 2004.-   Ohtake et al., Br. J. Cancer, 81:393-403, 1999.-   Orpana and Salven, Lymphoma 43:219-224, 2002.-   Park et al., J. Biol. Chem., 277:29730-29736, 2002.-   PCT Appln. PCT/US00/03745-   PCT Appln. PCT/US00/14667-   PCT Appln. PCT/US99/11913-   PCT Appln. PCT/US99/18441-   Peptide Synthesis, 1985-   Protective Groups in Organic Chemistry, 1973-   Protein NMR Spectroscopy, Principles and Practice, J. Cavanagh et    al., Academic Press, San Diego, 1996.-   Pulkkinen et al., Acta Otolaryngol., 117:312-315, 1997.-   Purushothaman et al., Blood, 115(12): p. 2449-57, 2010.-   Purushothaman et al., J Biol Chem, 283(47): p. 32628-36, 2008.-   Rajkumar et al., Clin Cancer Res, 6(8): p. 3111-6, 2000.-   Rajkumar et al., Clin Cancer Res, 8(7): p. 2210-6, 2002.-   Ramani et al., J Biol Chem, 287(13): p. 9952-61, 2012.-   Rapraeger and Ott, Curr. Opin. Cell Biol., 10(5):620-628, 1998.-   Rapraeger et al., FEBS J, 280(10): p. 2194-206, 2013.-   Rapraeger et al., J. Cell Biol., 103:2683-2696, 1986.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and    1570-1580, 1990.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 3:624-652, 1990.-   Ria et al., Leukemia, 17(10): p. 1961-6, 2003.-   Ridley et al., Science, 302(5651): p. 1704-9, 2003.-   Ridley, Cell, 145(7): p. 1012-22, 2011.-   Ritchie et al., Clin Cancer Res, 17(6): p. 1382-93, 2011.-   Roskelley et al., Curr. Opin. Cell Biol., 7:736-747, 1995.-   Sanderson and Bernfield, Proc. Natl. Acad. Sci. USA, 85:9562-9566,    1988.-   Sanderson and Borset, Ann. Hematol., 81:125-135, 2002.-   Sanderson and Yang, Clin Exp Metastasis, 25(2): p. 149-59, 2008.-   Sanderson, Semin. Cell Dev. Biol., 12:89-98, 2001.-   Sanz-Rodriguez et al., Br J Haematol, 107(4): p. 825-34, 1999.-   Schafmeister et al., J. Amer. Chem. Soc., 122(24): 5891-5892, 2000.-   Schnidrnaier et al., Int J Biol Markers 21: 218-222, 2006.-   Scudla et al., Neoplasma, 57(2): p. 102-10, 2010.-   Seidel et al., Blood, 95(2): p. 388-92, 2000.-   Singer et al., J. Cell Biol., 104:573-584, 1987.-   Solid Phase Peptide Synthelia, 1984-   Sotnikov et al., J Immunol, 172(9): p. 5185-93, 2004.-   Stanley et al., Am. J. Clin. Pathol., 112:377-383, 1999.-   Streeter and Rees, J. Cell Biol., 105:507-515, 1987.-   Sun et al., Int. J. Dev. Biol., 42:733-736, 1998.-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,597,457-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,790,421-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,889,155-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 6,093,573-   U.S. Pat. No. 6,261,569-   U.S. Pat. No. 7,183,059-   U.S. Pat. No. 7,192,713-   U.S. Patent Publication 2005/0015232-   Vacca et al., Am J Hematol 50: 9-14, 1995.-   Vacca et al., Br J Haematol, 87(3): p. 503-8, 1994.-   Vande Broek et al., Clin Exp Metastasis, 25(4): p. 325-34, 2008.-   Vlodavsky et al., Semin Cancer Biol, 12(2): p. 121-9, 2002.-   Wang et al., J Biol Chem 289: 30318-30332, 2014.-   Wang et al., J. Biol. Chem., 285:13569-13579, 2010.-   Weber, Advances Protein Chem., 41:1-36, 1991.-   Whiteford et al., J. Biol. Chem. 283: 29322-29330, 2008.-   Wider, BioTechniques, 29:1278-1294, 2000.-   Wiksten et al., Int. J. Cancer, 95:1-6, 2001.-   Woods and Couchman, Curr. Opin. Cell Biol., 13:578-583, 2001.-   Woods et al., Embo J., 5:665-670, 1986.-   Wu et al., J Immunol, 188(6): p. 2914-21, 2012.-   Yamashita et al., J. Immunol., 162:5940-5948, 1999.-   Yang et al., Blood, 100(2): p. 610-7, 2002.-   Yang et al., Blood, 105(3): p. 1303-9, 2005.-   Yang et al., J Biol Chem, 282(18): p. 13326-33, 2007.-   Yang et al., Mol. Cell Biol., 30(22):5306-5317, 2010.-   Zetser et al., Cancer Res, 63(22): p. 7733-41, 2003.

1. A method of inhibiting VLA-4 interaction with syndecan-1 comprisingcontacting VLA-4 and/or Syndecan-1 with a peptide segment consisting ofbetween 12 and 100 amino acid residues and comprising amino acidresidues 210-221, 210-233, 210-236, or 210-240 of SEQ ID NO:
 1. 2. Themethod of claim 1, wherein the peptide segment is 12, 13, 14, 15, 16,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 aminoacid residues in length.
 3. The method of claim 1, wherein the peptidesegment is between 12 and 50 amino acid residues in length.
 4. Themethod of claim 1, wherein the peptide segment is between 16 and 30amino acid residues in length.
 5. The method of claim 1, wherein thepeptide segment is between 23 and 27 amino acid residues in length. 6.The method of claim 1, wherein the peptide segment consists essentiallyof amino acid residues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO:4),210-236 (SEQ ID NO:2), or 210-240 (SEQ ID NO:3) of SEQ ID NO:
 1. 7. Themethod of claim 1, wherein the peptide segment comprises amino acidresidues 210-221 (SEQ ID NO:8), 210-233 (SEQ ID NO:4), 210-236 (SEQ IDNO:2), or 210-240 (SEQ ID NO:3) of SEQ ID NO:
 1. 8. The method of claim1, wherein the peptide segment consists of amino acid residues 210-221(SEQ ID NO:8), 210-233 (SEQ ID NO:4), 210-236 (SEQ ID NO:2), or 210-240(SEQ ID NO:3) of SEQ ID NO:
 1. 9. The method of claim 1, wherein theVLA-4 and/or syndecan-1 is located on the surface of a cell.
 10. Themethod of claim 9, wherein the cell is an endothelial cell or a lymphoidcell.
 11. The method of claim 9, wherein the cell is a cancer cell. 12.The method of claim 11, wherein the cancer cell is a carcinoma, amyeloma, leukemia, a melanoma, a lymphoma cell, a schwannoma, amalignant peripheral nerve sheath tumor cell, or a glioma.
 13. Themethod of claim 12, wherein the myeloma is multiple myeloma.
 14. Themethod of claim 12, wherein the leukemia is chronic lymphocyticleukemia.
 15. The method of claim 11, further comprising contacting thecancer cell with a second cancer inhibitory agent.
 16. The method ofclaim 11, wherein the cancer cell is a metastatic cancer cell or tumorstem cell.
 17. The method of claim 1, wherein contacting comprisesproviding an expression construct comprising a nucleic acid encoding thepeptide segment.
 18. The method of claim 9, wherein the cell isassociated with an inflammatory or autoimmune disease.
 19. The method ofclaim 18, wherein the cell is within a subject having an inflammatory orautoimmune disease, and whereby the inflammatory or autoimmune diseaseis treated.
 20. The method of claim 19, wherein the inflammatory orautoimmune disease that is treated is ulcerative colitis, Crohn'sdisease, rheumatoid arthritis, systemic lupus erythematosus (SLE), ormultiple sclerosis (MS).